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Chromatography

Liquid chromatography in combination with mass spectrometry (LC-MS) is an essential tool in the analysis of biological samples, especially in proteomics, metabolomics and other systems biology areas. Samples become increasingly complex and for contemporary bioscience research, LC-MS is therefore what the microscope was to early biologists.

Complex biological samples are challenging to analyze. Specialized sample preparations and separation techniques are traditionally needed to reduce the complexity. This leads to excessive sample consumption, augmented data variation and increased costs. The random structures of packed and monolithic columns causes peak dispersion and poor separation. The length of these columns is limited due to back-pressure build up. This hampers their ability to extract essential information.

An outstanding nano-LC separation using a PharmaFluidics µPACTM cartridge overcomes these challenges, resulting in less sample preparation, higher throughput and an unprecedented analytical result.

µPACTM – Pillar Array Columns

PharmaFluidics’ proprietary flow distribution structures are integrated in the µPACTM separation cartridges. Perfect flow distribution allows the transition from narrow in- and outlet tubing to much wider separation channels without introducing any significant peak dispersion.

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µPACTM separation beds are formed by carefully etching away the interstitial volumes out of a silicon wafer support, leaving an array of pillars. This creates a stationary phase support structure that is organized in a reproducible, perfectly ordered pattern.

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The perfect order of the etched bed eliminates peak dispersion introduced by otherwise heterogeneous (eddy) flows in disordered/packed beds, creating much higher plate counts and sharper peaks. Consequently, samples are diluted less during elution, increasing sensitivity. This enables the use of smaller sample volumes.

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Intrinsically free-standing, the pillar support structures allow flow with minimal back-pressure build up. As there are no obstructions by touching surfaces of the stationary phase, blocking the regular flow of the mobile phase, µPACsTM return low back-pressures at high flow rates.

With the separation bed being fixed to its silicon support, µPACTM cartridges can also be used in reverse flow mode.

µPACTM cartridges generate robust analytical data, with excellent repeatability and productivity.

Columns of unprecedented length (50 – 200 cm) are created on a small footprint. This is achieved by interconnecting several separation lanes by means of proprietary turn structures. Carefully engineered flow distributors allow to make these turns with no additional peak dispersion. The resulting long columns give the sample more time to interact with the stationary phase, leading to unrivalled separation power.

The µPACTM performance characteristics enable identification of many more components with less preparation and higher analytical throughput, also from minute samples.

PerformancePharmaFluidics µPACTM Chip cartridgeState-of-art HPLCStandard HPLC
Plate Number500.000 and up250.000150.000
Peak Capacity1.200 and up700300
Identifications10.000 and up5.0002.000
Sample Volumedown to 4 nl5.000 nl5.000 nl

Connectivity

According to the specific demands of your separation challenge, two distinct connection configurations can be provided.

Nanoliter sized sample volumes are recommended for all reversed phase chromatography applications where sample concentration is not a limiting factor. Moreover, precise definition of these sample volumes ensures minimal peak dispersion for early eluting molecules that have little hydrophobicity. 

In case of more diluted samples consisting of hydrophobic molecules, connection configuration compatible with microliter sized sample volumes can offer a suitable solution. In this configuration, sample volumes ranging from 1 to 20 microliter can be concentrated and subsequently separated on the µPACTM cartridge of your choice.

For small (nl range) sample volume injections a dedicated 4-port internal volume valve is used.  Herein the sample volume is determined in a small slit engraved in the valve’s internal seal (indexed ‘2’ and indicated by a white arrow).  Volumes currently available for this setup are 4, 10 or 20 nl.

LOAD/A position: sample flows over slit 2, while slit 3 ensures flow to the column. Slit 1 has no function in this position. INJECT/B position: slit 2, filled with sample, is introduced into the column flow. A volume equivalent to that of slit 2 is injected. Slit 1, inactive in the LOAD/A position, ensures that the sample path is not disrupted. Slit 3 on the other hand becomes inactive.

An injection setup with larger volume (µl range) injections is also possible. Opposed to small volume injections, the sample volume is determined externally in a loop connected to a standard 6-port valve. This setup offers a more versatile range of sample volumes as loops of different volumes can be used. Also dedicated sample injection programs with partial loop filling can be used to inject only a fraction of a specific loop volume. 

LOAD/A position: sample flows over slit 2, the external loop and slit 3. The external loop is filled with sample. Slit 1 ensures flow to the column. INJECT/B position: The external loop is introduced, via slit 1 and 2, into the column flow. A volume equivalent to that of the loop is being injected.  Slit 3 ensures the sample path is not disrupted.

Getting started

To serve your custom separation needs, µPACTM cartridges are available in various lengths and equipped with connectors matching your application. Stationary phase chemistry is currently focusing on reversed phase HPLC separations for applications in metabolomics and lipidomics.

Length

  • 50 cm
  • 100 cm
  • 200 cm

Stationary phase

  • C18 endcapped

Connectivity

  • 1-20 uL sample volume
  • 4-10 nL sample volume

However, the technology can be optimized for all types of analytes and new column types are in permanent development, expanding the area of focus to include applications for a broad range of industries, including Biotech, Bio-Pharma, Pharmaceuticals, Cosmetics, Food & Feed, Chemicals, Petrochemicals & Polymers, and Oil & Gas.

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