search.noResults

search.searching

dataCollection.invalidEmail
note.createNoteMessage

search.noResults

search.searching

orderForm.title

orderForm.productCode
orderForm.description
orderForm.quantity
orderForm.itemPrice
orderForm.price
orderForm.totalPrice
orderForm.deliveryDetails.billingAddress
orderForm.deliveryDetails.deliveryAddress
orderForm.noItems
41


pressure to run as the sub-2 µm particles [4]. To take full advantage of the fused core particle columns, traditional HPLC systems may need optimisation of fluidic flow paths and volumes, but the required pump pressure is typically available.


Development of µ-pillar array columns


Over the last three decades, a novel approach to manufacture separation columns has surfaced. Inspired by the original proposals by the group of Fred Regnier in the 1990s [5,6], investigations began into developing micro-machined pillar array columns. The µ-pillars can be positioned in perfect order within the separation channel, and act as the separation backbone. The potential for column-to-column repeatability is significantly higher than with randomly packed columns, just as the possible improvements in separation efficiency, with a factor of 2-3 in comparison to traditional packed-bed columns [7].


Furthermore, the µ-pillar structure leads to reduced flow resistance. Both the pillar diameter and pillar to pillar distance can be tightly controlled, not only resulting in homogeneous separation paths, but also opening the opportunity to create structures that can operate at much reduced back pressures, in comparison to sub-2 µm particle columns [8]. This provides opportunities for much longer columns, with examples reaching up to 4 x 200 cm coupled together, operating below its maximum pressure of 350 bar [9].


Technical background of µ-pillar array columns


In contrast to the rather stochastic slurry packing process that is used for packing of particles that possess an inherent size distribution in traditional columns - resulting in a somewhat random distribution of the particles within the flow path - the backbone of the stationary phase in micro-pillar array columns is designed, much in the same way as electronic circuits in a microchip are designed. This design, in the form of a photomask is used to reproduce the same geometrical pattern over and over again on silicon wafers using light, in a process called photo lithography. In short, a silicon wafer, covered with an appropriate hard mask material layer, is first covered with a photoresist [10]. Shining light through the photomask projects the geometrical pattern on the photosensitive material resulting


Table 1. µ-Pillar specifications


Separation length


µPAC™ 50 µPAC 200


50 cm 200 cm µPAC™ capLC 50 cm


µ-Pillar diameter


µ-Pillar height


Inter-pillar distance


Back pressure


5 µm 18 µm 2.5 µm 50 bar @ 300 nL/min 5 µm 18 µm 2.5 µm 100 bar @ 300 nL/min 5 µm 28 µm 2.5 µm 90 bar @ 5 µL/min


in a copy of the pattern after chemical development of the resist material. A dry etching step will subsequently remove the hard mask in those regions that are not protected by the remaining photoresist, making these regions accessible for the Deep Reactive Ion Etching (DRIE). This DRIE step removes silicon in the unprotected areas in a cyclic fashion, leaving perfectly vertical pillars and channel walls as defined by the photomask. In doing so, separation channels are formed that always contain the same number of pillars, are always located at the same well defined and perfectly ordered position, resulting in virtually identical copies of the same mask (typical variation both in position and dimensions ±50 nm) [11].


To achieve a higher loading capacity, the resulting micro structured wafers are subsequently rendered superficially porous using an electrochemical anodisation process in which the silicon wafer acts as the anode. Although the minimum plate height of about 5 µm hardly increases, the C-term increases significantly as the porous layer thickness increases [12]. With this in mind, a porous layer with a nominal thickness of 300 nm increases the loading capacity with a factor of 30 as compared to the non-porous case while keeping the C-term within acceptable levels [13]. After a few post-processing steps, the structured silicon wafer is anodically bonded to a glass wafer to obtain closed fluidic structures. As a last step, individual column chips are separated from the wafer-glass stacks by a process called dicing [14].


Individual µ-chips are transformed into chromatographic columns by first inserting and fixing the in- and outlet capillaries in the respective channels using a UV-curable gluing step, followed by an assembly step providing a protective housing and appropriate HPLC fittings. As a final step, the bonded phase is applied on the free surface of the pillars and the channel walls by coupling the bare backbones to a PharmaFluidics proprietary dedicated wet surface chemistry station.


The power of the µ-pillar array column technology lies in the fact that designs and realisations can be tuned to specific workflows based on the pillar shapes and diameters, pillar position and inter pillar distances and etching depth.


Application areas


In today’s laboratories, the µ-pillar array columns have found their way especially into omics and related research. The flow regime of the µ-pillar array columns can be divided into two distinct ranges, nanoLC at 50-2000 nL/min and capillaryLC at 1-15 µL/ min. With their extended lengths of up to 200 cm, the µ-pillar array columns match the requirements of omics applications that require highest sensitivity, resolution and reproducibility, and are ideal when only smallest sample volumes are available [15,16].


NanoLC is one of the gold standard separation techniques in proteomics allowing almost seamless connectivity to mass spectrometry (MS). Despite technological advances in MS detections and nanoLC systems, further developments of nanoLC columns has lacked a little. Connectivity to the nanoLC system has clearly improved, with the Thermo Scientific nanoViper connections as the prime example. Attempts to lower the volume from the column to the emitter have been pursued as well, e.g. PicoFrit® (New Objective) and EASY-Spray™ (Thermo Scientific), as well as chip-based separations like ionKey (Waters). But the traditional packed-bed separation channel has remained within all these formats, with the possible limitations as mentioned above.


This is where µ-pillar array columns can offer a significant step forward to further optimise the performance in low flow LC/MS. Taking full advantage of the previously described micro-machined lithographic manufacturing process and the tight control over the µ-pillar dimensions and position, the highest column to column reproducibility can be achieved. Figure 2 shows the reproducibility over seven µ-pillar array columns, separating from the same cytochrome c digest, with an average coefficient of variation of 0.63% over the nine peaks.


In addition, the µ-pillars are part of the original wafer where the separation channel is etched. In combination with the greatly reduced back pressures, as indicated in Table 1, this allows for a far higher number of sample injections than typically expected on a nanocolumn. Figure 3 shows a longevity experiment of µ-pillar array column, running for six months, performing the separation of


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76