subject: Short Note on the NMR Detection in Capillary Electrophoresis (CE) and Capillary Electrochromatography (CEC) [print this page] Short Note on the NMR Detection in Capillary Electrophoresis (CE) and Capillary Electrochromatography (CEC)
Short Note on the NMR Detection in Capillary Electrophoresis (CE) and Capillary Electrochromatography (CEC)
CHUEN-TAT, KANG ()
Individual Scientist and Researcher
Introduction to NMR Spectroscopy
NMR or Nuclear Magnetic Resonance has the ability to elucidate molecular structures, study molecular dynamics, measure molecular properties and bulk properties of the medium, allow analysed samples to be used for further investigation, determine impurities, study molecular binding and screen potential drug candidates due to its non-invasive nature.
NMR could be used as online detector when coupling with liquid chromatography (LC), according to Watanabe and Niki [1], of which the sensitivity latter improved by the development of better solvent suppression techniques, on-column sample pre-concentration, high field strength magnet and expanded flow cells.
NMR Microcoil
In NMR, the NMR is being applied to describe the active volume of 1 L or less. The signal-to-noise (S/N) ratio), related to the sensitivity and detection limit, is defined as the ratio of NMR resonance height to root mean square (RMS) noise. The S / N per unit volume for solenoid coils with diameter greater than 100 m is approximated by S/N b7/4 / dc [2] where b is the Lamor frequency.
The sensitivity of microcoil probe could be described with a volume independent parameter known as mass sensitivity (Sm) : Sm = ( S / N ) / (mol t1/2) where mol and t are number of moles within Vobs and the acquisition time, respectively.
Electrophoretic Current Induced and Flow Effects
Solenoid coils from the most mass sensitive RF coils with orthogonal orientation to the static magnetic field (Bo). The strength of this induced magnetic field (Bi) is Bi = oir / (2R2) where o is the permeability constant, i is the electrophoretic current, R is the capillary i.d. and r is the radial distance [3].
In addition to induced magnetic field, the different migration rate can greatly influence the NMR signal intensity and line width. Under static conditions, the effective longitudinal (T1eff) and transverse relaxation (T2eff) times of NMR nuclei in a flow system can be related to the residence time of the analyte () in the detector and relaxation times (T1, T2) under static conditions [4] :
1 / T1eff = 1 / T1 + 1 / 1 / T2eff = 1 / T2 + 1 /
Applications of CE / CEC-NMR
Capillary electrophoresis (CE) and capillary electrochromatography (CEC) has been used to separate simple mixture to more complicated biofluidic mixture [6].
In CE-NMR, chip-based unit with microfabricated microcoils may be able to analyse picoliter volume samples [7]. The separation efficiency can be improved by optimizing the buffer pHs.
CE-NMR is able to analyse simple amino acid mixture like glycin, cysteine and argenine according to its first report [5]. The potential application of CE-NMR and CEC-NMR for analysis of metabolites in biofluids has been demonstrated [8]. CE-NMR has successfully analysed the major metabolites of paracetamol in human urine [6]. Comparison of chemical shift for two major metabolites, paracetamol sulfate conjugates and paracetamol glucuronide, as well as endogeneous material that has been characterised, has confirmed the presence of these compounds. With S / N of 3, the estimated amount that can be detected ~ 10 ng.
The instrumentation required for CEC-NMR has added benefit that its sample loading capability is higher, making detection easier than conventional CE-NMR although the instrument used is almost the same. According to Gfrorer P et al. [9] gradient elution, which is a widely used technique to achieve faster and / or better separation of complex mixture, is a powerful technique that is able to successfully analyse an analgesic mixture containing caffein, acetylsalicylic acid and acetaminophen. In order to increase the separation efficiency, a large sample injection volume is required. The total separation time has been reduced by one-third and the separation has been improved significantly by applying a solvent gradient of 0% to 30% CD3CN in 25 min with the solvent gradient in comparison to isocratic solution. Unfortunately there are adverse effects, namely chemical shift change and line broadening, from a solvent gradient elution on NMR spectral. These can be obstacles in performing steep solvent gradient with online NMR detection although these effects do not interfere with spectral interpretation in this work.
When pressurized capillary electrochromatography (pCEC) coupled to NMR, mixture of unsaturated fatty acid esters could be separated and identified and this has proven that applied pressure during CEC can decrease the separation time [10].
NMR Thermometry in CE and CEC
Several NMR chemical shifts in fact could be used to probe the temperature of samples due to its temperature sensitivity [11]. Lacey et. Al. introduced CE / NMR thermometry to measure intracapillary temperature [12]. This is based on the concepts that intracapillary temperature could alter the pH of the buffer, pKa, peak shapes, overall efficiency and migration times in electrophoretic measurements [13] and the events like DNA / RNA separation, aggregation, protein confirmation and denaturing are affected by temperature. Factors like water O-H stretching frequency [14], absorption spectrum of Co (II) chloride [15], buffer conductivity [16], electroosmotic mobilities [16] and miscellar capacilty factor [17] could be used to calculate the internal temperature of CE.
According to Lacey [12] in the study of intracapillary temperature evolution in CE as a function of time at different voltage. The temperature remains constant prior to voltage application, where low voltage such as 2 kV cause only small increase in temperature (~ 1C) even after 15 minutes of temperature application but latter the temperature will reach a higher steady state value, the rate of increase of temperature is greater for higher voltage.
Future Direction
One suggestion to make CE-NMR and CEC-NMR to become more widely applied technique is to increase the sensitivity of NMR. Possible solutions to use multiple RF coils on a single column, so that NMR spectral of a single migration band are recorded at multiple points. Fluidic systems containing multiple coils that allow individual bands to be parked and probed with NMR while continuing the separation and higher-field-strength magnets will continue to become available, increasing the performance of NMR. The approach like the separation and identification of molecular classes using pattern recognition can minimize long analytical procedures involved in metabolomics and proteomics that could be performed accordingly. The appearance of applications using capillary scale separations hyphenated to NMR is expected to increase greatly over the coming decades.
References
[1] Watanabe N, Niki E. Proc Jpn Acad Ser B 1978, 54. 194-199.
[2] Peck TL, Magin RL, Lauterbur PC. J. Magn Reson Ser B 1995.
[4] Albert K, Bayer E. High performance liquid chromatography proton nuclear magnetic resonance on-line coupling. In : patonay G, ed. HPLC Detection, New York : VCH, 1992 : 197-227.
