Liquid chromatography (LC or high-performance liquid chromatography, HPLC) is the most common chromatographic method used in bioanalysis. HPLC is used to separate analytes due to their chemical properties mostly in particle packed or monolithic columns.
Normal particle packed analytical columns have an inner diameter (ID) of 1.0 to 4.6 mm.
When particle packed columns are miniaturized the packing process becomes more vulnerable as it can produce unfavorable small voids which deteriorate chromatographic separation. Therefore the need for perfect and homogenous packing is emphasized. The same challenge comes up with monolithically packed columns, where especially the batch-to-batch, repeatability is not necessarily high. Furthermore, all voids and dead volumes after the HPLC column reduce the column separation efficiency. A weak point for separate electrospray tips and HPLC columns comes from the interface between them, as the junction can give large dead volumes relative to the low flow rates. Therefore HPLC columns, which are integrated to the same microchip with an ESI emitter, provide better performance in principle.
Efforts to miniaturize HPLC columns focus on reduction of the inner diameter and extra-column dispersion sources of particle or monolithically packed extra-columns in order to decrease flow rates, increase separation efficiency and lower detection limits.41,42 Kutter et al. (2000) reviewed the possibilities and challenges of capillary electrophoresis in miniaturization, which was the first trend in miniaturization of separation channels.43 CE is the most common technique applied to microchip separations, because it is easy to miniaturize as it needs only a narrow channel for the separation, and the electrodes needed
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are easily fabricated on the microchip. For microchip HPLC columns there is a need for high pressure and to date there are not many genuinely working microchips made which are combined with MS. Tomer et al (1994), characterized the effect of miniaturization in HPLC where decreasing the inner diameter of the column by 100-fold increased the relative concentration at a detector over 8000-fold (Table 1.1).44 The larger the column the higher stability and loading capacity of the column is obtained, whereas better sensitivity is achieved with the decrease of the column ID. Therefore, the choice of column dimensions is always a compromise between capacity and sensitivity.
Table 1.1. Characteristics of HPLC columns, adapted from Tomer et al.44
Column ID* Volume Flow rate Injection
The first article on particle packed 0.5 – 1.0 mm ID stainless steel capillaries was published in 1967,45 and they can be considered the first miniaturized particle packed columns when compared to conventional analytical HPLC columns with 3.0 – 4.6 mm ID.
Later, at the beginning of the 1980s, development of capillary LC columns was intensified.46,47,48 Varga et al. published a review about miniaturization for proteomics research in 2003.49 A year later, peptide separations with monolithically filled capillaries and a microcolumn with an integrated ESI nib were published.50 The microchip was fabricated from SU-8 polymer and a monolithic stationary phase with C12 functionality was used for reversed-phase separations, however no chromatogram of microchip separations was presented. One of the first miniaturized HPLC columns combined with an integrated nanoESI source and an enrichment column with good performance was published in 2005 (Fig. 1.9).51 The microchip was fabricated of laminated polyimide layers where channels were patterned using laser ablation technology. The drawback of using laser ablation is that each microchip has to be fabricated separately, increasing fabrication time and costs. The microchip consisted of an integrated injector, a sample enrichment column, a separation column, and a nanoESI tip. A typical flow rate on the microchip was 100 – 400 nL/min and the high voltage needed ESI was 2.4 kV. This microfluidic integration of nanoLC components allowed subfemtomole detection of tryptic digestion products. This microchip is also commercialized by Agilent Technologies (HPLC-Chip).
In 2005, Xie et al. published an integrated reversed-phase column with frits for bead packing, an ES nozzle, electrolysis based gradient pumps, and a low volume mixer (Fig.
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1.10).52 The microchip was fabricated on a silicon wafer using parylene and SU-8 polymers. The separation column was 12 mm long and packed with C18 coated particles.
A flow rate of 80 nL/min was used for separations. The microchip was applied to an LC/MS analysis of a mixture of peptides from trypsin digestion. The microchip showed similar gradient separation when compared to commercial nanoflow LC separations. The flow control with integrated gradient pumps was challenging and therefore separations were difficult to get repeatable.
a) b)
c)
Figure 1.9. a) A polyimide microLC-ESI microchip. The dark pattern on the right end of the chip is the electrodeposited metal for contact to the fluid flow channel near the ES tip. b) Schematic of the chip rotor interface shown in the LC run mode. The chip (brown) has ports (light blue) leading to the sample enrichment column (yellow) and to the LC separation column (brown). The rotor (black) has channels (red) that rotate with respect to the chip. c) (top) Extracted ion chromatograms of four ions (m/z 395.5, 547.7, 582.7, and 499.7) and (bottom) base peak chromatogram of 20 fmol BSA digest at 100 nL/min.51
In conclusion, only a few publications of integrated ES tips together with miniaturized HPLC columns have been published. The main challenge of the integrated microLC columns comes from the high pressure needed, thus making the production of tight fluidic connections and microchips that can tolerate high pressures critical. Furthermore, in order to obtain functional LC columns the packing procedure should be homogenous and reproducible with minimal dead volumes.
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a) b)
c)
Figure 1.10. a) A photograph of a microfluidic LC-ESI microchip. b) A diagram of the LC-ESI microchip showing the placement of the solvent reservoir and cover plate on top of the main chip.
c) Comparison of the extracted ion chromatograms for eight tryptic peptides from BSA separated using the microchip LC (left panel), and an Agilent 1100 series HPLC system (right panel). For the Agilent run, gradient formation started at 2 minutes. For the chip LC run, gradient formation started at time 28 minutes.52