Hadamard transform capillary electrophoresis

This paper reports the first demonstration of a multiplex sample injection technique in capillary electrophoresis. The sample was injected into a capillary (effective length, 4 cm) as a pseudorandam Hadamard sequence by a photodegradation technique using a high-power gating laser, and the fluorescence signal, which was measured using a probe excitation beam, was decoded by an inverse Hadamard transformation. The signal-to-noise ratio was improved by a factor of 8, which was in good agreement with the theoretically predicted value of 8.02. This approach is potentially useful for the enhancement of the sensitivity by 3 orders of magnitude in high-resolution capillary electrophoresis, combined with fluorescence detection.     

Ultratrace analysis based on Hadamard transform capillary electrophoresis

Hadamard transform capillary electrophoresis, which is based on a multiple sample injection technique, was combined with laser-induced fluorometry and utilized in the determination of analytes at subpicomolar levels. The sensitivity was substantially improved by increasing the order, i.e., the number of elements, of the Hadamard matrix. In fact, the signal-to-noise ratio was enhanced 18-fold by the use of a matrix of order 2047. A feasibility study was carried out by computer simulation to study the detection of an average of less than a single molecule in a single injection volume. The signal peak was clearly observable even under conditions at which only 0.3 molecule is present in the volume. Thus, this approach is potentially useful for ultratrace analysis, in which conventional single-injection capillary electrophoresis cannot be applied.     

Hadamard transform microchip electrophoresis combined with diode laser fluorometry

The application of a Hadamard transform technique to microchip electrophoresis is described. The sample is electrokinetically injected into a separation channel and is then detected by diode laser-induced fluorometry. The sample and buffer solutions are introduced into the channel by controlling the high voltages applied to the solutions, according to a code determined by a Hadamard matrix. The S/N ratio of the signal in the electropherogram can be improved by a factor of 5 in comparison with that obtained by a conventional single-injection method, although an 8-fold improvement is theoretically predicted when a 255-order matrix is used.     

Fast Hadamard transform capillary electrophoresis for on-line, time-resolved chemical monitoring

We report a new approach for collecting and deconvoluting the data in Hadamard transform capillary electrophoresis, referred to as fast Hadamard transform capillary electrophoresis (fHTCE). Using fHTCE, total analysis times can be reduced by up to 48% per multiplexed separation compared to conventional Hadamard transform capillary electrophoresis (cHTCE) while providing comparable signal-to-noise ratio enhancements. In fHTCE, the sample is injected following a pseudorandom pulsing sequence derived from the first row of a simplex matrix (S-matrix) in contrast to cHTCE, which utilizes a sequence of twice the length. In addition to the temporal savings provided by fHTCE, a 50% reduction in sample consumption is also realized due to the decreased number of sample injections. We have applied fHTCE to the analysis of mixtures of neurotransmitters and related compounds to yield improved signal-to-noise ratios with a total analysis time under 10 s. In addition, we demonstrate the capability of fHTCE to perform time-resolved monitoring of changes in the concentration of model neurochemical compounds.     

Hadamard transform ion mobility spectrometry

A detection scheme that makes use of the Hadamard transform has been employed with an atmospheric-pressure ion mobility spectrometer fitted with an electrospray ionization source. The Hadamard transform was implemented through the use of a linear-feedback shift register to produce a pseudorandom sequence of 1023 points. This pseudorandom sequence was applied to the ion gate of the spectrometer, and deconvolution of the ion signal was accomplished by the Hadamard transform to reconstruct the mobility spectrum. Ion mobility spectra were collected in both a conventional and Hadamard mode, with comparisons made between the two approaches. Initial results exhibited low spectral definition, so an oversampling technique was applied to increase the number of data points across each analyte spectral peak. The use of the Hadamard transform increases the duty cycle of the instrument to 50% and results in a roughly 5-fold enhancement of the signal-to-noise ratio with a negligible loss of instrument resolution. It is also shown that any potential multiplex disadvantage, which limits the attractiveness of some high-throughput techniques, is not a limiting factor in this new implementation.     

Hadamard transform ion mobility spectrometry

Traditionally, the spectrum acquired using ion mobility spectrometry (IMS) is an average of multiple experimental cycles. Each cycle is initiated by passing a short burst of ions into a drift tube containing a homogeneous electric field. Prior to starting the subsequent cycle, all ions in the system must arrive at the detector or spectral overlap may occur. To maximize resolution, the ion pulse admitted to the drift tube is small in relation to the total scan time with the unfortunate consequence of an inherently low duty cycle (approximately 1%). Offering an improved SNR through a 50% duty cycle, the Hadamard transform (HT) applied to ion mobility spectrometry represents a fresh alternative to signal-averaged data acquisition. Initial results from measurements of amphetamine and cytochrome c samples indicate a 2-10-fold increase in SNR for the HT-IMS technique with no reduction in resolution.     

Fourier transform capillary electrophoresis with laminar-flow gated pressure injection

Fourier transform capillary electrophoresis (FTCE) was developed as a method to improve signal-to-noise ratio (S/N) and resolution in capillary electrophoresis (CE) separation. In FTCE, multiple simultaneous CE separations were performed in the same channel system and interrogated using a single-point detector. To illustrate experimentally the improvement offered by FTCE in S/N ratio and resolution, we carried out a modest number (five) of multiple injections and separations. We show even with this small number of separations, S/N increased by a factor of 2.9, and theoretical plate height improved by a factor of more than 30. We demonstrated this technique with laser-induced fluorescence detection, but a wide variety of detection methods are compatible with FTCE.     

Comparison of Hadamard transform and signal-averaged detection for microchannel electrophoresis

A Hadamard transform (HT) detection method for microchip capillary electrophoresis with laser-induced fluorescence and a charge-coupled device (CCD) is described and compared to signal-averaged detection. A low-noise CCD camera is used to image a section of a separation channel where each camera pixel can be thought of as a unique detector. For signal averaging, electropherograms corresponding to individual pixels can be averaged for improved S/N. HT detection is performed on each pixel electropherogram to generate a contour plot electropherogram. The multiple injections required for HT provides an enhancement at the cost of longer times for the pseudorandom injection sequences. A short sample injection length of 0.25 s is used to reduce the overall analysis time and improve sensitivity compared to previously published results. An injection sequence is performed on the microchip that is based on a cyclic S-matrix of 513 elements that generates an 8-fold improvement in S/N compared to a single injection. This spatially resolved HT detection method is also capable of performing a multicomponent separation. Signal-averaged HT and single-injection data are compared to experimental HT and single-injection results. The unique capabilities of each method are described.     

On-line fourier transform infrared detection in capillary electrophoresis

The coupling of Fourier transform infrared (FT-IR) spectroscopy as a new on-line detection principle in capillary electrophoresis (CE) is presented. To overcome the problem of total IR absorption by the fused-silica capillaries that are normally employed in CE separations, a micromachined IR-transparent flow cell was constructed. The cell consists of two IR-transparent CaF2 plates separated by a polymer coating and a titanium layer producing an IR detection window, 150 microm wide and 2 mm long, with a path length of 15 microm. The IR beam was focused on the detection window using an off-axis parabolic mirror in an optical device (made in-house) attached to an external optical port of the spectrometer. The connections between the fused-silica capillaries and the flow cell were made by a small O-ring of UV-curing epoxy adhesive on the sharply cut ends of the capillaries, allowing the capillaries to be easily replaced. Aqueous solutions comprising mixtures of adenosine, guanosine, and adenosine monophosphate were used to test the system's performance. Conventional on-line UV detection was employed to obtain reference measurements of analytes after the IR detection flow cell. The limit of FT-IR detection for all analytes (in absolute amounts) was in the nano- to picogram range corresponding to concentrations in the low-millimolar range.     

Dating silk by capillary electrophoresis mass spectrometry

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 μg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.     

Fourier transform atomic absorption flame spectrometry with continuum source excitation

The design and performance of a Fourier transform atomic absorption flame spectrometer (FT-AAS) is presented. A 300-W xenon arc continuum source and a Michelson interferometer are used. A signal to noise disadvantage arising from the multiplex feature of FT-AAS is demonstrated by varying the photon flux at the detector without changing the exciting radiation. A grating is used for dispersion of the radiation before the interferometer to reduce the spectral window at the photomultiplier tube. Detection limits for several elements are generally an order of magnitude poorer than those obtained by continuum atomic absorption methods using echelle-grating spectrometers. Line profiles and absorption spectra, within the region of the spectral window selected by the grating, can be obtained with this method. Standard curves for sodium were constructed to extend the linear calibration range, by using absorbances measured at the absorption maximum and 0.022 nm off-line.     

Hadamard transform time-of-flight mass spectrometry: a high-speed detector for capillary-format separations

This work demonstrates that with an intrinsic duty cycle of 50% and spectral storage speeds up to 277 spectra s(-1) Hadamard transform time-of-flight mass spectrometry (HT-TOFMS) is a promising detector for any capillary-format separation that can be coupled to MS by electrospray ionization. Complete resolution of the components of a nine-peptide standard was achieved by coupling pressurized-capillary electrophoresis (pCE) to HT-TOFMS. The addition of pressure to the separation capillary decreased analysis times and stabilized the electrospray ionization source. Pulsed-pressurized injection of reserpine was used to experimentally simulate narrower peaks than those obtained in the pCE. HT-TOFMS was able to sample peaks having widths in the millisecond range.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

A metal nebulizer capillary electrophoresis/Fourier transform infrared spectrometric interface

A capillary electrophoretic (CE) system has been successfully interfaced to a Fourier transform infrared spectrometer. The advantage of such an interface is that analytes may be detected and often unequivocally identified without analyte derivatization. The interface consists of a stainless steel tube in which the CE capillary is placed and the two are held in contact with the use of a metal tee. A solvent elimination approach is used with the interface, so that analytes are deposited onto an infrared transparent window, that is, CaF2, and measured with the use of an infrared microscope. A critical component of this design is to provide an electrical connection at the end of the CE column to permit stable separations that allow for efficient transport of the sample onto the window. The interface produces an aerosol that is directed at the surface of the infrared transparent window. The use of a volatile electrolyte, along with the flow of helium, allows for partial evaporation of the electrolyte in flight and complete evaporation of the solvent and electrolyte on the surface of the window to produce a "dry", or neat, analyte deposit.     

On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis

Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development.     

Characterization of a capillary zone electrophoresis/electrospray-mass spectrometry interface

A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fused-silica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary ("electrodrilling") can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.