140 with acetonitrile/water solvent mixture. In some examples, the sample is diluted at least 2 times, at least 2.5 times, at least 3 times, at least 5 times of its volume. After diluting the sample 140, the sample is injected 150 onto a hydrophilic interaction liquid chromatography column, which can be connected to a liquid chromatography system. In some embodiments, at least one component of the hydrophilic interaction liquid chromatography column and liquid chromatography system, e.g., Waters™Arc™ Premier System for LC available from Waters Corporation, Milford, MA, 01757, comprises a fluid-contacting alkylsilyl coating on a metal surface (e.g., flow path).

At least one component of the liquid chromatography system may be selected from several components including a fluid manager system (e.g., controls mobile phase flow through the system), tubing (which could also be replaced or used together with micro fabricated fluid conduits), fluid connectors (e.g., fluidic caps), frits, a chromatography column (e.g., XBridge Premier column available from Waters Corporation, Milford, MA, 01757), a sample injector including a needle to insert or inject the sample into the mobile phase, a vial, sinker, or sample reservoir for holding the sample prior to injection, a detector, such as a mass spectrometer, and a pressure regulator for controlling pressure of the flow. Interior surfaces of the components of the chromatographic system form a fluidic flow path that has wetted surfaces.

At least a portion of the wetted surfaces can be coated with an alkylsilyl coating. The coating can be applied by vapor deposition. As such, methods and devices of the present technology provide the advantage of being able to use high pressure resistant materials (e.g., stainless steel) for the creation of the flow system, but also being able to tailor the wetted surfaces of the fluidic flow path to provide the appropriate hydrophobicity so deleterious interactions or undesirable chemical effects on the sample can be minimized. In some examples, the coating of the flow path is non-binding or low-binding or non-interactive with respect to the analyte, such as oligosaccharides with DP 6 or higher.

The alkylsilyl coating can be provided throughout the system from the tubing or fluid conduits all the way through to the detector, e.g., UV detector. The coatings can also be applied to portions of the fluidic path (e.g., at least a portion of the fluidic path). That is, one may choose to coat one or more components or portions of a component and not the entire fluidic path. For example, the internal portions of the column and its frits and end caps can be coated whereas the remainder of the flow path can be left unmodified. Further, removable/replaceable components can be coated. For example, the vial or sinker contained in the sample reservoir can be coated; other replaceable components, such as frits, can be coated as well.

In general, the alkylsilyl coating is applied through a vapor deposition technique. Precursors are charged into a reactor in which the part to be coated is located. Vaporized precursors react on the surfaces of the part to be coated to form a first layer of deposited material. The vapor deposition can be applied in a stepwise function to apply a number of layers of deposited material to the surfaces to grow a thickness of the coating and/or to apply layers of different materials (e.g., alternating between a first and second material) to form the coating.

Further information regarding the coating and the deposition of fluid-contacting coatings in accordance with the present technology is available in US 2019/0086371, which is hereby incorporated by reference. US2019/0086371 also describes tailoring of a coating to meet the needs of a particular sample/analyte.

In some embodiments, the fluid-contacting alkylsilyl coating is made using vapors of bis(trichlorosilyl)ethane, bis(trimethoxysilyl)ethane, bis(trichlorosilyl)octane, bis(trimethoxysilyl)octane, bis(trimethoxysilyl)hexane, and bis(trichlorosilyl)hexane.

In some embodiments, at least a portion of the wetted surfaces of the flow path are coated with multiple layers of the same or different alkylsilyls, where the thickness of the alkylsilyl coatings correlate with the number of layering steps performed (e.g., the number of deposited layers of alkylsilyl coating on wetted surface of the flow path of the chromatographic system/device). In this manner, increasingly thickness of the fluid-contacting coatings can be controlled and tailored to achieve desirable separations.

In some embodiments, the alkylsilyl coating is formed from one or more of the following precursor materials bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. Other embodiments of alkylsilyl coatings suitable for use with the present technology are described in U.S. Patent Publication No. 2019/0086371 (see, in particular Table 1) and U.S. Application Publication No. 2022/0118443 (both of which are hereby incorporated by reference). For example, the alkylsilyl coating can include or consist of a C2 coating, which is the product of vapor deposited bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

In some embodiments, prior to coating, all metal components are passivated according to a nitric acid passivation. Passivated parts and a silicon wafer are then introduced to the vapor deposition chamber and vacuum is established. In some embodiments, the first step is a 15-minute, 200 Watt, 200 cc/min oxygen plasma cleaning step, and the next step is the first vapor deposition cycle. In some embodiments, each vapor deposition cycle contains a silane vapor deposition, followed by the introduction of water vapor for silane hydrolysis. In one example, the silane vapor is delivered at a pressure of 2.0 Torr for 5 seconds, and then the water vapor is delivered at a pressure of 50 Torr for 5 seconds. Following delivery, the silane and water is left to react with the substrate for 15 minutes. This cycle is repeated to produce the desired number of layers and coating thickness.

After injected the sample onto a hydrophilic interaction liquid chromatography column 150, the injected sample is flowed through the column 160, and separated with the hydrophilic interaction liquid chromatography 170. The separated sample is then passed through a detector to be analyzed. In some embodiments, the detector is a UV detector and/or a fluorescence detector. In certain embodiments, multiple detectors can be used. For example, the UV detector and/or a fluorescence detector can be couple to a mass spectrometer.

METHODS AND EXAMPLES
Example 1: Comparative Examples for 2-AB Labeled Dextran Ladders with DP Values 2 to 11

Using the methods described above, dextran ladders with DP values that varies from 2 to 11 were separated in systems using uncoated hardware and coated hardware in accordance with the present technology. In each of the examples, the comparative chromatograms demonstrate that consistent and high responses have been obtained for dextran ladders having DP 6 or higher using the Premier Glycan BEH Amide column (with alkylsilyl coating). Inconsistent and low responses (for DP≥6) were observed when the conventional Glycan BEH Amide column (without coating) was used.

The samples including dextran molecules were labeled with 2-AB molecules, and HILIC separation of 2-AB labeled dextran ladders were performed under the conditions recommended in AOAC Official Method 2021.01 (available from AOAC International, Rockville, MD).

Standard samples were prepared at 1 mL in aqueous solution for analysis. First, 250 μL water was added to 500 μL dextran solution. Then, the internal standard (laminaritriose) was added to the sample. After taking 20 μL solution from this dextran-internal standard solution, 100 μL water and 100 μL 2-AB labeling reagent were added. The solution was mixed in water bath at 65° C. for 1 hour. After 1 hour, 1 mL of acetonitrile/water solution (75/25 v/v) was added to the mixture prior to analysis. Analysis conditions are provided below.

Methods:

- LC system: ACQUITY H-Class Plus (QSM) with ACQUITY FLR Detector. Arc™ Premier System (BSM) with ACQUITY FLR Detector
- Column: XBridge™ Glycan BEH Amide Column, XBridge™ Premier Glycan BEH Amide Column, 2.5 μm, 2.1×150 mm.
- Col. temp.: 25° C.
- Inj. Vol.: 2 μL
- Run time: 60 min.
- Mobile phase A: Acetonitrile
- Mobile Phase B: 100 mM ammonium formate pH 4.4
- FLR Detector: Excitation λ=330 nm; Emission λ=420 nm
- Elution program: (see Table 1 below)

TABLE 1

Elution Program

% B

Time
Flow
% A
(Ammonium formate

(min)
(mL/min)
(Acetonitrile)
100 mM, pH 4.4)

0
0.6
88.0
12.0

7.0
0.6
88.0
12.0

17.0
0.6
85.0
15.0

21.0
0.6
85.0
15.0

36.0
0.6
72.6
27.4

44.0
0.6
54.0
46.0

44.1
0.3
54.0
46.0

44.5
0.3
30.0
70.0

49.5
0.3
30.0
70.0

52.0
0.3
88.0
12.0

54.0
0.6
88.0
12.0

60.0
0.6
88.0
12.0

FIG. 2A-FIG. 2B show a comparison of 2AB labeled dextran ladder chromatographs on traditional standard columns (i.e., uncoated columns and system tubing etc.) (FIG. 2A) and coated Premier columns (i.e., columns and systems coated in accordance with the present technology) (FIG. 2B). Difference in relative peak height found for oligosaccharides with DP 6 and higher using the Premier Glycan BEH Amide column compared to the conventional Glycan BEH Amide column shows that the responses obtained in coated column and system is higher compared to the conventional column and system. For example, peak height for DP 8 for the coated column is about 5 EU, whereas the DP 9 peak for the traditional uncoated column is only about 4.8 EU.

FIG. 3 shows relationship of dextran relative peak area (relative to the internal standard) obtained on coated (Premier) and traditional standard BEH Glycan Amide columns (uncoated). FIG. 3 shows how the injections on a traditional column show inconsistent and low response starting from DP 6 (dextran glucose unit 6). In contrast, the first and eighteenth injection on the Premier column show the same results proving consistency of the method. The responses were normalized to an I.S. (laminaritriose). The same sample (containing an IS and dextrans with different DP) was used in the comparison.

FIG. 4 shows dextran response on standard uncoated columns, proving loss of analytes after DP 6 or higher. As DP increases, analyte loss increases for these uncoated columns.

FIG. 5A and FIG. 6A show chromatograms of a dextran ladder (containing DP 2 to 11 and an internal standard) after injected to a traditional column (i.e., no coating) and traditional system (FIG. 5A) and a coated column and coated system (FIG. 6A). FIG. 5B and FIG. 6B show chromatograms of a solvent blank immediately after running the dextran ladder in the traditional column and system (FIG. 5B) and in the coated column and system (FIG. 6B). While FIG. 6B shows no carry-over (i.e., no measurable carry-over) for dextran ladders with DP 6 or higher, FIG. 5B shows carry-over (measured as 4%) from dextran ladders with DP 6 or higher in the same region of the chromatogram, proving the positive effect of coating on the elimination of carry-over of dextran ladder with DP 6 or higher.

Carry-over results in different systems were investigated. Carry-over is recognized as the presence of small analyte peaks that appear when a blank is injected following the injection of a sample that produces large peaks. When it occurs, peaks attributed to the previously analyzed sample may be observed in the subsequent chromatogram(s) which may co-elute or interfere with desired analytes. Percent carry-over is calculated by the following formula:

% carry-over=(carry-over peak area(s)/internal standard peak area)×100%.

While around 4% carry-over (FIG. 5B) was observed after running dextran ladders with DP 6 or higher on non-Premier column (uncoated) connected to a non-Premier system (uncoated, ACQUITY H Class PLUS), only 0.2% carry-over was observed when the same samples were run on non-Premier column (uncoated) connected to a Premier system (coated, Arc Premier System). No carry-over was observed when samples were run on a Premier column (coated column) connected to a Premier system (coated, Arc Premier System) see