Regular capillary of a Fida Instrument is made of glass (silicon dioxide O=Si=O) and carries negative charges on its surface (silanol group -SiOH). When a glass surface encounters an aqueous solution, ionization occurs which leads to negative surface charge. Many proteins and especially DNA/RNA binding proteins carry net positive charges and therefore are electrostatically attracted to glass surfaces.

Background information

Dynamic coating of a capillary can alleviate adsorption of analytes to the surface of the capillary. These coatings can modify the negative charge of the silanol groups (-SiOH) responsible for adsorption. The coating should be stable and non-interfering with analyte. There are three different coating agents: amines, polymers, and surfactants. Examples of amine are monoamine (hydroxylamine, ethylamine), diamines (cadaverine, putrescine, agmatine), polyamines (spermidine, spermine). Polymers can be cationic or anionic.Examples of cationic polymers are polybrene, poly (diallyl dimethylammoniumchloride) (PDADMAC), polyarginine (PA), cationic agarose, chitosan, poly (ethyleneoxide) (PEO), hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose(HEC), PVA, and poly (N, N-dimethylacrylamide) (PDMA). Chitosan, cationicamylopectin derivatives, cellulose has also been used as dynamic coating for analysis of DNA, Examples of surfactants are cetyltrimethylammoniumbromide (CTAB) for anion analysis. The main role of cationic surfactants in BGEis reversal of the EOF and suppression of interactions between cationicanalytes and the inner surface of the capillary wall. We can use single or double-chain surfactants, anionic, zwitterionic, and cationic surfactants, or amixture of cationic and zwitterionic, which may result in micellar layerformation or bilayer coating on the inner surface of the capillary. This is strictly connected with the CMC—it must be one half to one third of the CMC.

Procedure for dynamic coating

Materials

- Dynamic HS coating reagent (310-050)
- MillQ water, NaOH (1 M)
- Standard Capillary (100-001)
- Vial set, pkg of 200 pressure vials(including caps and inserts) (210-200)

Instrument:

Fida Neo and 480-detector.

Procedure:

I. Launch Fida Neo data acquisition software and place the tray 2 in sample load positions, either by opening and closing the door or by going into the manual control and choosing sample load under inlet positions and pressing apply (Figure 1).

Figure 1: Manual Control, Inlet Position set to sample load
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II. Prepare the following solutions:

- 1 M NaOH, I mL
- Milli Q water
- Dynamic HS coating reagent

Prepare two vials with no inserts (Figure2). Pipette 1 mL of NaOH and milli-Q water into two separate vials.
Label them, snap the top tightly and insert them in tray-2 in positions 1 and 2 respectively,
according to the tray 2 vial map in Coating Procedure method (Figure 3).

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Figure 3: Coating procedure method. Note the position of NaOH, milli Q water
and coating solutions 1, 2, 3 respectively.

Take a one vial of dynamic HS coating solution and unscrew the cap and remove the insert which contains the coating solution. You might need a pair of tweezers to remove the insert. Once you remove the HS coating solution, place it in an empty vial, label it, snap a pressure cap tightly and insert it at position 3 on in tray 2. Close tray door.

III. In FIDA software:
1. Under ''method'', load the coating procedure. Notice the positions for NaOH (1 M), H₂O, and coating solution (1, 2 & 3). Coating procedure starts with an NaOH rinse which will clean and strip away any previous coating, followed by water flush, coating and water flush again. Each step is 300 seconds long and is done at 3500 mbar.

2. Click sequence, and then click add a line or double click in the first row of cells to generate the first assay. By default, the software will generate an assay with generic indicator name with indicator and analyte positions at 1 and 11 and waste position at 1 in triplicates, using standard capillary method.

Figure 4: Sequence for dynamic coating. Note that only one replicate is needed.
Analyte and indicator positions are left as default as they are specified in the method.

3. Double click on the first cell under method and scroll down and select coating procedure. If you do not find the coating procedure there, select browse and find it from the list of methods.

4. Under replicate, change from 3to 1. We do not need to repeat this method as each method will strip the coating by running NaOH.

5. Under sample name, double click and change the sample name into coating. Since coating procedure uses only tray2, and there are no analyte or indicator, there is no need to modify thosecells. You can leave them as they are.

6. Under folder, select a location to save the coating results.

7. Click “Start sequence”. It should take about 21 minutes for the coating procedure to finish.

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