Report
Membrane Preconcentration CE he chromatographic separation of complex mixtures prior to detection is a rich and varied field of study. Numerous techniques, including GC, LC, and supercritical fluid chromatography, have been developed and described. More recently, CE has found widespread use as a powerful technique, particularly for separating complex mixtures derived from biological matrices. CE separations are typically carried out in narrow bore (5-100 um i.d.) fused silica capillaries. A high voltage (2-30 kV) is applied across the capillary and analytes are separated to afirstapproximation based on their charge-to-mass (z/m) ratios (1) This separation technique is characterized by low solvent consumption (nanoliters to microliters per analysis) and high theoretical plate value equivalents (—1-2 x 106). Furthermore, sample losses are minimized because of the limited surface area and volume (—1-2 uL) of the capillary. However, limited surface area and volume mean limited loading capacity, resulting in poor concentration limits of detection (CLOD) This problem has proven to be a major limitation of CE in the analysis of biologically derived analyte mixtures (2) Resolution and separation efficiency are usually compromised when sample injection volumes of less than 1 5—2T of the total capillary volume are used when using conventional capillaries onlvfinitevolumes of -1-100 nL can be iniected Efforts have been made to improve poor CLOD by on-line preconcentration prior to separation (2). Most groups pursuing this approach have used a 1-2 mm bed of solid-phase packing material at the Qing Y a n g Andy J . T o m l i n s o n S t e p h e n Naylor Mayo Clinic and Foundation
A new approachtopreconcentratingsamples before separation. inlet end of the CE capillary (4-10), a microvariation of the classical solid-phase extraction cartridge. However, CE performance is compromised with such an approach (4-6,11) because a relatively large bed volume is needed at the inlet, which results in increased back pressure and reduced hydrodynamicflowwithin the CE capillary (4,11). In addition, the ion flow is also impaired, resulting in a reduced or anomalous electroosmotic flow (EOF), which causes erratic analyte migration times. Furthermore because relatively large volumes of organic solvent are required to analytes from the solid phase peak broadening compromised resolution and loss of separation occur (12) Finallv construction of solid-phase cartridges is poor because of the difficulties encountered in producine- consistent bed volumes and packintrs of the solid
phase materials. To overcome the limitations of the solidphase cartridge, we replaced the bed of solid phase with a defined polymeric mem-
brane impregnated with a chromatographic stationary phase (e.g., C2, C4, C8, C18, mixed phase, and ion-exchange). Furthermore, we have developed analytical conditions for performing membrane preconcentration with CE (mPC-CE) (13,14). In this Report, we describe the development of mPC-CE and mPC-CE with MS (mPC-CE/ MS). We also detail how this approach is used describe how conditions are established to optimize analytical performance, and give specific examples. Constructing the cartridge
To construct the mPC-CE cartridge, the membrane adsorptive phase is installed in a cartridge that is usually prepared from Teflon tubing, as shown in the middle of Figure 1 (13). A piece of membrane is cut out using a 22-gauge blunt-tipped hypodermic needle. The membrane in the needle is inserted into the midpoint of a short length (—1 cm) of Teflon tubing (300 um i.d. x 1500 um o.d.). The needle is placed at one end of the Teflon tube and, with a small length of fused silica positioned inside the
Analytical Chemistry News & Features, March 1, 1999 183 A
Report hypodermic needle, the membrane is carefully pushed into position. Provided this procedure is performed carefully, the piece of membrane will hold its shape and completely fill the cross-section of the Teflon tube. The final step is to insert two fused silica capillaries onto each end of the Teflon tube. During this process, care is taken to neither compress the membrane nor scrape the walls of the Teflon tubing, which could block the cartridge and lower the hydrodynamic flow. The tight fit between the inside of the Teflon and the outside of the fused silica affords a leak-free cartridge with a push-fit connection. The push-fit cartridge is advantageous because, if the piece of membrane becomes heavily contaminated, it is easily replaced by simply unclipping the entire cartridge from the CE capillary. Prior to installation, the membrane is activated by
washing with methanol and then CE separation buffer. The cartridge is connected to the CE separation capillary by a piece of 1-cm-long polyethylene tubing, which tightly holds the outlet end of the cartridge and the inlet end of the CE capillary with the tips of the two capillaries positioned as close to each other as possible. The entire mPC-CE capillary is then conditioned under high pressure (20 lb/in2) with separation buffer for 10 min in the CE instrument. Subsequent sample loading washing and elution also carried out under high To perform mPC-CE, analytes are loaded directly onto the cartridge in either the original physiological fluid or a mixture of buffer and solvent. The impregnated membrane phase is selected to afford high-affinity adsorption of analytes. After the analytes have been adsorbed
and concentrated, the membrane is washed with CE separation buffer to remove any salts and other debris. Ultimately, analytes are eluted from the membrane with a minimum of organic or aqueous/organic solution, and subsequently, the CE voltage is applied to affect analyte separation. A major advantage of the membranebased preconcentration approach is the minimized bed volume of the adsorptive phase, which makes it possible to reduce the volume of elution solvent required for efficiently removing analytes. This simple change, coupled with reduced hydrodynamic flow and reduced ion impedance during electrophoresis through the mPC-CE cartridge, results in a more reproducible EOF (12,15). Furthermore, the high adsorptive capacity of the impregnated membranes permits the loading and analysis of large volumes (10-800 uL) of dilute solutions without unduly compromising either analyte resolution or separation efficiency afforded bv conventional methods In addition the reproducible conmembrane bed volume t o runrnrliir'ible anahTte mim-Qtion timf>c
However the use of larire loading volumes of analyte solutions can lead to some com promiseri CE erformflnce (?) h'ch h avoided by carefully selecting post-eluuon conditions that effectively cause either analyte stacking or focusing on applying the voltage across the CE capillary. Analyte stacking and transient isotachophoresis
Figure 1 . Schematic of mPC-CE. The middle shows an exploded view of the mPC-CE cartridge. (a) Off-line sample loading of the mPC-CE cartridge using a pressurized bomb, (b) On-line mPC-CE/MS with electrospray ionization using a coaxial liquid sheath configuration.
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In the case of small organic molecules such as drug metabolite and toxin mixtures, relatively large volumes (60-150 nL) of organic elution solvent are required to ensure high analyte recovery from the membrane. Furthermore, when such organic molecules are eluted from the membrane in methanol and/or CH3CN, a phenomenon known as analyte stacking occurs when the CE voltage is applied (1,16, 17). Stacking (Figures 2a and b) occurs because the analytes are now in a lowconductivity solution zone relative to the CE separation buffer which leads to a high field strength through the organic solvent and results in rapid migration of the analytes through this zone
Analytes in a conventional CE capillary are stacked either at the leading edge if they are positively charged or at the trailing edge if negatively charged (2,18). This is shown in Figure 3a for the mPC-CE separation of a mixture of the drug haloperidol and five structurally similar putative metabolites. Although 55 uL of a solution containing 3.3 ng/mL of each compound was loaded onto the mPC cartridge, baseline resolution of all six compounds was achieved using analyte stacking in conjunction with mPC-CE To efficiently remove biopolymers such as peptides and proteins from the adsorptive membrane, an organic elution solvent that contains some water (1030%) is necessary (2,12,15,18.. Furthermore, optimal biopolymer recovery is achieved only when less than 50 nL of such solvent mixtures are used (12). Using a relatively large volume of elution solvent with some water in it results in inefficient analyte stacking and, consequently, some peak broadening and loss of analyte resolution. However using moving boundary transient isotachophoresis (tITP) after analytes have been eluted from the membrane restores normal CE performance (19) In this case tITP conditions are used to stack analytes zone
