Reagent Bulletin for Post-column Liquid Chromatorgaphy 20

o-Phthalaldehyde For Automated Post-column Derivatization of Primary Amines Here you can download this materials in PDF format* INTRODUCTION Primary amines form highly fluorescent adducts when reacted with o-Phthalaldehyde (OPA) and a mercaptan under basic conditions. At a pH>9 and ambient temperature, reaction is generally complete within 30 seconds. The products of this reaction, 1-alkyl-2-alkylthio-substituted isoindoles, exhibit optimal excitation at 330 nm and maximal emission at 465 nm. The reagents described below are intended for postcolumn derivatization of primary amines such as: amino acids in native samples and protein or tissue digests those formed of carbamate herbicide those derived from other sources.

REAGENT COMPONENTS

o-Phthalaldehyde,
Chromatographic Grade™

Pickering’s OPA is specially prepared to meet the demanding requirements of highsensitivity pre- and post-column HPLC derivatization methods. Our repurification process actually begins with commercially available 99% material.

The entire process is controlled to prevent trace amine contamination. Lot quality is verified by post-column HPLC using a high-sensitivity fluorescence detector.

Thiofluor™,
Chromatographic Grade™

Pickering's Thiofluor, a solid, low-odor nucleophile, is a superior substitute for 2-Mercaptoethanol in the preparation of OPA reagents. It forms a more stable reagent and longer-lasting fluorophore with OPA than does 2-Mercaptoethanol, yet it has the same fluorescence properties.

Unlike the volatile 2-Mercaptoethanol, Thiofluor will not migrate through the gas manifold and regulator of the OPA reagent pressurization system.

Two grams of Thiofluor is equivalent to 1 mL of 2-Mercaptoethanol.

o-Phthalaldehyde Diluents, Chromatographic Grade™

Three application-specific diluents are available from Pickering Laboratories: CB910 and GA104 for carbamate and glyphosate analyses, and OD104 for amino acid analysis.

These borate solutions are produced from starting materials which are free of heavy metals and amines. As with most other products of Pickering Laboratories, the quality of the OPA Diluents is verified by actual post-column HPLC analysis.

REAGENT PREPARATION

Storage and Handling:

The prepared reagent is sensitive to both light and oxygen. It should be formulated in a container which can serve as the instrument reservoir, such as the 1L reservoir assembly included with the Pickering post-column instrument. Always store, transfer, and use the prepared reagent under an inert gas (N₂, He, Ar). Stored under inert gas in the glass reagent reservoir, the reagent will maintain its original signal-to-noise ratio for one week or more.

OPA Reagent for Carbamate Insecticide Analysis

1. Pour 950 mL of o-Phthalaldehyde Diluent (Cat. No. CB910) into the reservoir. Save 5 mL for Step 5.

2. Thoroughly de-aerate the contents by sparging with the same inert gas used to protect the reagent when it is in the instrument. Bubble the gas through a gas dispersion tube (frit) at 3 L/ minute for a minimum of 10 minutes.

3. To 100 mg of o-Phthalaldehyde (Cat. No. O120) in a clean, dry container add 10 mL of HPLC grade Methanol. Mix until homogeneous.

4. Remove the gas dispersion tube from the solution. Add the mixture to the deoxygenated Diluent in the reservoir. Wash any residual mixture into the reservoir with an additional 1–2 mL of Methanol.

5. Dissolve 2 g of Thiofluor in the reserved 5 mL of the degassed OPA diluent and add into the reservoir.

Note: Once the reagent is mixed, securely capped and under inert gas pressure, do not bubble gas through the reservoir.

OPA Reagent for Glyphosate Herbicide Analysis

Pour 950 mL of o-Phthalaldehyde Diluent (Cat. No. GA104) into the reservoir.

• Continue with steps 2–5, above. Preparation of OPA Reagent for Amino Acid Analysis Pour 950 mL of o-Phthalaldehyde Diluent (Cat. No. OD104) into the reservoir.

• Continue with steps 2–5, above, except use 300 mg OPA.

• Add 3 mL of 30% aqueous Brij 35®†. Stir slowly or swirl gently to avoid foaming. When homogeneous, the reagent is ready for use. Brij 35 is necessary to prevent fluorescence quenching of the Lysine and Histidine derivatives.

REAGENT RESERVOIR CONNECTIONS

If the reagent is to be used in a Pickering Laboratories post-column instrument, the proper reservoir assembly, pump, and post-column connections will be in place.

The post-column system must be equipped with a means of maintaining a reagent reservoir pressure of 2 to 4 psi with an inert gas, typically N2 . The tubing employed to connect the reservoir to the gas regulator, and to connect the reservoir to the pump must be air impermeable. For this reason, Saran®†† tubing is recommended over fluorocarbon (e.g., FEP) and polyolefin (-ethylene, -propylene) tubing. The tubing between the reagent pump and the mixing tee should be 1/16 inch OD x 0.030 inch ID Type 316 stainless steel or 1/16 inch OD x 0.020 inch ID PEEK.

NOTE: DO NOT USE fluorocarbon tubing with an air-sensitive reagent!

† A 30% Brij 35 solution is available from Fisher Scientific Co., Cat No. BP 345-500.
†† Saran has approximately 1/1000 the oxygen permeability. Typical values at 23° C are: Saran, 0.9cc/mil/100in2; 986cc/mil /100in2.

Chromatographic Grade is a trademark of Pickering Laboratories, Inc.
Thiofluor is a trademark of Pickering Laboratories, Inc.
Saran is a registered trademark of Dow Chemical Corporation.

ORDERING INFORMATION

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