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Some Applications of the Electrospray Ionization System

Davis et al. [18], used an Electrospray Ionization system to couple a microbore column to a Finnigan MAT TSQ 700 triple sector Quadrupole Mass Spectrometer. The basic layout of the tandem system is shown in figure 27. The microbore column was 15 cm long, 0.25 mm I.D. and packed with a C18 reversed phase support. The flow rate was only 1-2 μl /min and, thus, a special electrospray assembly was designed to accommodate these low flow rates. The gradient was performed and stored in the manner of Snyder and Saunders [19] and Katz and Scott [20]. The eluent was monitored by a UV detector, and the exit flow from the detector passed to the micro electro-jet assembly. The micro-spray was constructed from a flame-drawn length of fused silica tubing, 5 cm long, 350 μm O.D. and 150 μm I.D. The aperture diameter of the drawn jet ranged from 1-5 μm.


Figure 27 The On-line Micro-Electrospray Ionization System Used with a Microbore Column

Two lengths of 150 mm O.D. and 25 mm I.D. tubing were placed inside the tube, and sandwiched between them was a hydrophilic PVDF frit.

Figure 28. Comparative Chromatograms of a Cytochrome c Standard Digest Mixture Monitored by UV Absorption and by the Base Peak Intensity

These insert tubes reduced the dead volume of the system, and provided a support for the filter, which was essential to avoid the jet becoming blocked. Even with this precaution, however, the tube life was limited, and could become blocked after only a few hours of use.

The jet was positioned accurately by means of a micrometer-driven optical rail assembly. Spray potentials ranged from 500-1000 V, and the optimum operating potential appeared to be about 100 V in excess of that at which spray droplets started to form. The micro-electrospray microbore column arrangement was used to monitor the separation of a number of polypeptides and an example of the separation obtained form 2 pmol of sample is shown in figure 28.

It is seen the chromatograms are similar, and little or no loss of chromatographic resolution takes place in the micro-electrospray system. The sample size was 2 pmol (if a mean molecular weight of 1000 is assumed, this will represent a sample mass of about 2 ng). However, a better example of the sensitivity obtainable from the system is afforded by the reconstructed chromatograms shown in figure 29. This sample size was only 40 fmole, (again assuming a mean molecular weight of about 1000, would be equivalent to a sample mass of 40 pg).

Figure 29. Selected Mass Chromatograms for Three of the peptide s from a Sample of Lys C Digest of Cytochrome

It is seen that the combination of the microbore column and the micro-electrospray provides a very high sensitivity without compromising the performance of either the liquid chromatograph or the mass spectrometer.

Another interesting application not only demonstrates the efficacy of the device for natural product investigation, but also shows some of the foibles that can be associated with the liquid chromatography column. Due to the increase in contemporary popularity of herbal remedies in the United States, there has been a need for analytical techniques to quality control imported substances, to ensure their integrity and safety. Van Breeman et al. [21], developed a method for measuring the ginsenoside content of ginseng products, marketed as roots, capsules, tablets and liquid extracts. ginsenoside s consist of a series of triterpine saponins in proportions that are typical of their country of origin. The individual ginsenoside s can be separated by reverse phase chromatography and ion exchange chromatography, but the use of specialized carbohydrate columns, containing aminopropyl functional groups, have also proved useful. Van Breeman et al., examined the ginseng root powder by extracting the powdered root with aqueous methanol , evaporating to dryness, dissolving the residue in water. They extracted the ginsenoside s by passing the solution through a solid-phase extraction tube.

A. Ginsenoside standard added by direct infusion. B. ginsenoside standard introduced from liquid chromatograph C. ginsenoside extract introduced from liquid chromatograph.

Figure 30. Electrospray Mass Spectra of ca 100 pmol of Ginsenoside

The retained material was displaced with butanol, evaporated to dryness, and redissolved in methanol . Samples of the methanol solution were placed on a carbohydrate analysis column (aminopropyl bonded to silica ) and separated using a water/acetonitrile gradient. As the ion monitored was an adduct of sodium (M+Na)+, the mobile phase also carried 100 μ M sodium chloride. The column eluent was monitored by a standard Hewlett-Packard 5989B MS engine Quadrupole Mass Spectrometer, and the spectra that were obtained for a specific ginsenoside are shown in figure 30.

The standard sample, injected directly into the mass spectrometer, gave the expected sodium adduct ion, (M+Na)+ at an m/z value of 970. However, it is seen that two new peaks appears at m/z values of 1067 and 1085. When the root extract is separated and monitored under the same conditions, there is no peak at 907 m/z and the peaks at m/z values of 1067 and 1085 have become much bigger, and that at 1085 is now the major peak. Although all standard ginsenoside sample produced both the sodium adduct ion and the [M+138]+ ion, the natural products only gave the [M+138] +.

It was also found, that the 138 adduct to the ginsenoside was most likely to be the protonated adduct (3-aminopropyl)-trihydroxysilane, [NH3(CH2)3Si(OH)3]+ which was present either as a reagent contaminant in the bonded phase or was produced by the decomposition of the stationary phase. The 1067 ion appeared to be produced by the removal of water from the [M+138]+ ion. The spectra represented only 100 pmol of ginsenoside and so the appearance of the (3-aminopropyl)-trihydroxysilane adducts might be a function of the sample size and if larger charges were employed the sodium adduct might again appear.

The size of the droplets is vital in electrospray techniques and Wilm and Mann [22] pointed out the lower the flow rate the electrospray, the smaller the droplets produced. Small droplets have high surface to volume ratios, and, thus, make a large proportion of the analyte molecules available for desorption. The authors fabricated capillaries with spraying orifices having only 1-2 μm I.D which could accommodate flow rates as little as 20 nl/min. The droplet diameter was claimed to be about 200 nm, in comparison with 1-2 μm, the diameter of droplets generated from the normal electrospray sources. As a result, the droplet volume was also 100 to 1000 times smaller and the miniaturized electrospray inlet could be operated without a sheath flow or pneumatic assistance, which significantly simplified the design of the interface. The modified form of the electrospray sampling system was not employed with an LC/MS interface, but with a MS/MS tandem instrument. An example of a Spectrum of ovalbumin obtained using the interface is shown in figure 31.

The Spectrum was taken from a 1 μl sample of an aqueous solution of ovalbumin containing 5 pmol/μl, and so it was confirmed that the interface functioned well with aqueous solvent mixtures. Assuming a molecular weight of about 44,000 for ovalbumin , this sample volume contains a mass of about 0.2 μg of protein at a sample concentration of about 0.02%. The Spectrum is the result of the deconvolution of the aggregate of 19 scans. The resolution obtained at this molecular weight was about 1300.

Figure 31. The Mass Spectrum of Ovalbumin Analyzed Using the Modified Electrospray Source.

An interesting extension of the utilization of this interface is its use with a MS/MS tandem instrument. The results published by Wilm and Mann, are shown in figure 32. The top curve shows the mass Spectrum of the peptide over the m/z range of 600 to 850. Two ion adducts from the preliminary ionization at about m/z values 670 and 810 were chosen for subsequent examination, and the results obtained are shown by the two spectra in the lower part of the figure 32.

Figure 32. The MS/MS Spectra from a Mass Separation of a Mixture of peptides.

It is seen that whereas the sample adduct ions had m/z values of 670 and 810, the m/z range of the MS/MS spectra extend to m/z values of 1100 and 1500. One explanation for this difference might be that the original adduct ions having m/z values of 670 and 810 carried multiple charges. It would appear that the sensitivity limits of the electrospray interface can vary widely from one design to another, and also, perhaps, between one type of sample and another.

Figure 33. An LC/MS/MS Combination with Post-column Metal Chloride Addition Using a Triaxial Electrospray Probe.

A technique using on-line post-column adduct formation in an electrospray inlet system was developed by Kohler and Leary [23] for the analysis of carbohydrate mixtures. A triaxial electrospray probe was designed that would allow the introduction of the sample solution, the nebulizing gas and a metal chloride reagent solution simultaneously into the probe jet. A diagram of their apparatus is shown in figure 33.

Two LC pumps provided gradient elution facilities and the sample was placed on the column with a low dispersion sample valve, having a 5μl loop. After injection, the sample, contained in the mobile phase, passed through a 0.5 μm filter onto the column. A diagram of the triaxial probe is shown in figure 34.

Figure 34. The Triaxial Electrospray Probe

The center tube carried the mobile phase from the column, the next coaxial tube carried the reagent, and the outside coaxial tube provided nitrogen to assist the nebulization. The post-column addition of metallic chlorides to a mobile phase carrying carbohydrates provides greater sensitivity, and assists in structural elucidation. The relative abundance of the protonated carbohydrate and the metal complexes with lithium , sodium , potassium , rubidium and cesium are shown in figure 35.

Figure 35. The Relative Abundance of the Protonated Carbohydrate and the Metal Complexes with Li, Na, K, Rb and Cs

The results shown in figure 35 reveal that the use of alkali metals complexes increases the sensitivity of the system to the carbohydrate, resulting from the enhanced ionization in the electrospray ionizing system. The lithium complex is shown to be over seventy times more abundant than the protonated species. Although the lithium complex appears to provide the maximum sensitivity, later, but separate experiments, indicated that cobalt complexes could give even greater sensitivity.

Employing the triaxial electrospray with cobalt chloride as the complexing reagent, the chromatogram of 1 nmol of a mixture of different carbohydrates, produced by single ion monitoring, is shown in figure 36.

Figure 36. The Separation of Four Oligosaccharides Using Metal Chloride Post-column Reagents

Although the chromatographic separation was not complete, the single ion monitoring of the cobalt complex ions clearly shows the presence of each carbohydrate.

The use of different reagents to enhance ion production in Electrospray Ionization has been of considerable general interest and the subject of a number of investigations It was employed by Van Breeman [24] in the analysis of carotenoids . Carotenoids have been shown to be the metabolic precursors of vitamin A, and, in addition, are also thought to have anticancer activity, and to act as in vivo antioxidants.

Van Breeman examined the use of the post-column injection of halogenated compounds, to enhance ionization efficiency. The ionization efficiency of several different halogen compounds were examined, including chloroform, 2,2,3,3,4,4,4-heptafluoro-1-butanol, 2,2,3,3,4,4,4-heptafluoro-1-butyric acid, 1,1,1,3,3,3-hexafluoro-2-propanol and trifluoroacetic acid. The relative effect of the different additives on the efficiency of the positive ion electrospray production was examined, and the results that were obtained for the different halogen compounds are shown in figure 37.

HFBA=2,2,3,3,4,4,4-heptafluoro-1-butyric acid, TFA trifluoro acetic acid, HFP=1,1,1,3,3,3-hexafluoro-2-propanol and HFB=2,2,3,3,4,4,4-heptafluoro-1-butanol

Figure 37. The Efficiency of Different Halogenated Compounds for Enhancing Ion Production when Employing Electrospray Ionization

The solute used in the experiments was β-carotene and 0.25 μg was injected onto the column. It is seen that the presence of the halogen antioxidants can significantly affect the ionization efficiency. However, it is also clear that an excess of the additive can have the opposite effect and reduces the ionization efficiency. It is seen that the 1,1,1,3,3,3-hexafluoro-2-propanol provided the greatest sensitivity. The limits of detection for both β-carotene (at an m/z value of 536 and lutien (at an m/z value of 568) are shown in figures 38 (A) and 38(B) respectively.

Figure 38. The Limits of Detection for β-Carotene and Lutein Employing 2,2,3,3,4,4,4-Heptafluoro-1-Butanol as an Antioxidant.

Sensitivities of about 2 pmol and about 0.6 pmol for β-carotene and lutein, respectively, are shown to be obtainable.


The results from the application of the same technique to the determination of carotenoids in heat processed canned sweet potatoes using the reagent heptafluorobutanol is shown in figure 39,

Figure 39. Positive Ion Electrospray LC/MS Analysis of an Extract of Heat Processed Canned Sweet Potatoes

(A) to (E). (A) shows the computer reconstructed mass chromatogram of the β-carotene molecular ion, at an m/z value of 536, from the injection of ca 20 ng of extract. (B) shows the computer reconstructed mass chromatogram of the ion at an m/z value of 568, which corresponds to lutein. (C) shows the computer reconstructed mass chromatogram of the ion at an m/z value of 552, displying the isomers of β-cryptoxanthin. (D) shows the chromatogram provided by the output of the Diode Array UV detector at 450 nm, recorded during the analysis shown in (A). (E) shows the chromatogram provided by the output of the Diode Array UV detector at 450 nm, recorded during the separation of 2 μg of sweet potato extract. The addition of 2,2,3,3,4,4,4-heptafluoro-1-butanol, post-column, as an ionizing enhancer has increased the overall sensitivity to carotenoids by about two orders of magnitude.

Sodium replacement ions can also be used to identify the charged state of the molecular ion. This possibility was used by Neubauer and Anderegg [25] to identify the charged states of peptide ions, when employing LC/MS and an electrospray ionization system. The presence of sodium acetate at sub-millimolar levels in the mobile phase, promotes the formation of sodium replacement ions in addition to the normally observed protonated species. The m/z spacing of the sodium adducts provide an unambiguous identification of the charged state of the ions and hence their actual mass. It was also found that at the necessary sodium concentration (ca. 250 μM) the sodium salt did neither interfere with the chromatographic process, nor cause undue fouling in the mass spectrometer ion source.

The effect of different amounts of sodium acetate in the mobile phase on the pattern of ion peaks produced in the mass spectra, is shown in figure 40. It is seen that as the quantity of sodium acetate in the mobile phase is increased, the number of sodium replacement ions also increases. The data shown in chromatogram (D) in figure 40 was used to identify the charge on the molecular ion of the peptide .

Figure 40. Electrospray Mass Spectra of a peptide Employing sodium Adduct Ions to Help Identify of the Charged State of the Molecular Ion

The m/z values for the five sodium replacement ions were plotted against the number of sodium atoms in the respective replacement ion, and curve is shown in figure 41.

Figure 41. Graph of m/z Values against Number of sodium Atoms in the Replacement Ion

The result is a straight line, with an index of determination of 1.000. The slope of the line is 11.06, which is about half the atomic weight of the sodium atom and, consequently, the ions must be doubly charged. Taking the doubly charged peptide ion to have m/z value of (674.6-1), (Peptide + H+), the actual molecular weight of the peptide will probably be

(674.6-1) x 2 = 1347.

Ionizing agents added, pre-column and post-column, can be used in a number of interesting and useful ways to augment the basic information provided by the mass spectrometer.

To eliminate the restrictions imposed by the nature of the solvent on the function of the electrospray ionizing system, Banks et al. [26] developed an ultrasonically assisted nebulizer, to allow aqueous mixtures of nucleosides to be separated and monitored by a LC/MS tandem instrument, using the electrospray interface. The interface they developed is shown in figure 42.

 


Figure 42. The Electrospray Interface Fitted with an Ultrasonic Nebulizer

Except for the ultrasonic nebulizing unit, the interface was similar to those already described. It consisted of a 0.005 in. I.D., 1/16 in. O.D., stainless steel tube, which had been ground to a sharp point at one end, and then fitted into a two-part stainless steel body with a nut and ferrule. A pair of piezoelectric crystals was fitted between the two stainless steel parts, and was driven by a function generator and a power amplifier. The device was designed so that there was independent control of the needle potential (V(needle)), the cylindrical electrode potential, (V(cyl.), the nosepiece potential, (V(nose)), and the capillary entrance potential, (V(ent)). In the layout shown in figure 42, the needle was always maintained at ground potential and the temperature of the drying gas was held at 82˚C. In other respects, the system closely resembled the standard type of electrospray interface, typically described by Yamashita and Fenn [27]. It was found that for effective nebulization, the ultrasonic vibrator frequency must be adjusted to the resonant frequency of the device, which was identified by experiment. The optimum frequency had to be carefully controlled, as a deviation of 0.1 kHz at 180 kHz could reduce the spray efficiency by nearly two thirds. The sample used for optimizing the conditions of ion production was adenosine dissolved in pure water (100 pmol/μl), which was considered a 'worst case' example. An example of the use of the interface in the separation of some nucleosides , using a microbore column and a Hewlett-Packard mass spectrometer model HP88A, is shown in figure 43.

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1. cytidine, 2. uridine, 3. 5-methylcytidine, 4. 2-O-methylcytidine, 5. 1-methyladensonine, 6. guanosine , 7. 2-O-methylguanosine, 8. N2-methylguanosine, 9. adenosine and 10. N2-N2-dimethylguanosine.

Figure 43. The Total Ion Current Chromatograms of the Separation of Some nucleosides

The sample contained the nucleosides from a tRNA digest, reduced to its substituent nucleosides by the combined action of nuclease P1, and bacterial alkaline phosphatase . The sensitivity of the device is illustrated by the results shown in figure 43. The interface operates well with liquids having high water contents, and in fact, operates well when nebulizing samples in pure water. It is noted, however, that very small samples exhibit a disproportional loss of minor components, which may make quantitative assays uncertain when operating at maximum sensitivity. The electrospray ionizing system has rendered the mass spectrometry available to a wide range of applications in biology and biochemistry, which hitherto, were precluded due to its inability of other ionizing systems to function efficiently with aqueous solutions.

Hua et al. [28] employed Electrospray Ionization in their examination of brevetoxins . Brevetoxins are produced by the dinoflagellate , Gymnodinium breve, and are responsible for killing fish and also pose certain health risks to humans. brevetoxins were isolated from an extract of cultured material using a reversed phase solid-state extraction procedure. A microbore column was employed as the separation vehicle and the eluent was split at a T-junction, part passing to a UV detector (215nm) and the remainder to the mass spectrometer (Vestec Model 201 fitted with an electrospray inlet system). The Electrospray Ionization source was modified by replacing the nozzle (the counter electrode) by a flat stainless steel plate pierced by a 0.44 mm in diameter hole. The 200 l /min pump, normally used to evacuate the first stage of the ion source, was replaced with a 500 l /min. pump. These modifications reduced the lower detection limit by a factor of four. The results obtained from a test mixture of brevetoxins are shown in figure 44. The upper chromatogram was obtained from the UV detector and those below by total ion monitoring and selective ion monitoring respectively. The sample consisted of 1 μl of a solution, containing 20 ng/μl (a mass of 20 ng) and the mobile phase eluent was split in the ratio 3:1, to the UV detector and the spectrometer respectively. The peaks correspond to sodium adduct ions of the respective brevetoxins .

Figure 44. The Analysis of a Mixture of Brevetoxins Employing an LC/MS Tandem Instrument with a Modified Electrospray Interface.

Although the Electrospray Ionization source is one of the more popular LC/MS interface more recently, another type of ionization source that operates at atmospheric pressure, is now a strong competitor.


 

About the Author
RAYMOND PETER WILLIAM SCOTT was born on June 20 1924 in Erith, Kent, UK. He studied at the University of London, obtaining his B.Sc. degree in 1946 and his D.Sc. degree in 1960. After spending more than a decade at Benzole Producers, Ltd. Where he became head of the Physical Chemistry Laboratory, he moved to Unilever Research Laboratories as Manager of their Physical Chemistry department. In 1969 he became Director of Physical Chemistry at Hoffmann-La Roche, Nutley, NJ, U.S.A. and subsequently accepted the position of Director of the Applied Research Department at the Perkin-Elmer Corporation, Norwalk, CT, U.S.A.
In 1986 he became an independent consultant and was appointed Visiting Professor at Georgetown
University, Washington, DC, U.S.A. and at Berkbeck College of the University of London; in 1986 he retired but continues to write technical books dealing with various aspects of physical chemistry and physical chemical techniques. Dr. Scott has authored or co-authored over 200 peer reviewed scientific papers and authored, co-authored or edited over thirty books on various aspects of physical and analytical chemistry. Dr. Scott was a founding member of the British chromatography Society and received the American Chemical society Award in chromatography (1977), the M. S. Tswett chromatography Medal (1978), the Tswett chromatography Medal U.S.S.R., (1979), the A. J. P. Martin chromatography Award (1982) and the Royal Society of Chemistry Award in Analysis and Instrumentation (1988).
Dr. Scott’s activities in gas chromatography started at the inception of the technique, inventing the Heat of Combustion Detector (the precursor of the Flame Ionization Detector), pioneered work on high sensitivity detectors, high efficiency columns and presented fundamental treatments of the relationship between the theory and practice of the technique. He established the viability of the moving bed continuous preparative gas chromatography, examined both theoretically and experimentally those factors that controlled dispersion in packed beds and helped establish the gas chromatograph as a process monitoring instrument. Dr. Scott took and active part in the renaissance of liquid chromatography, was involved in the development of high performance liquid chromatography and invented the wire transport detector. He invented the liquid chromatography mass spectrometry transport interface, introduced micro-bore liquid chromatography columns and used them to provide columns of 750,000 theoretical plates and liquid chromatography separations in less than a second. Dr. Scott has always been a “hands-on” scientist with a remarkable record of accomplishments in chromatography ranging from hardware design to the development of fundamental theory. He has never shied away from questioning “conventional wisdom” and his original approach to problems has often produced significant breakthroughs.

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