HPLC Derivatization and Visualization Reagents

p-Bromophenacylate Reagent (p-bromophenacyl bromide) (0.1 mmol/mL) (Thermo Scientific™)

Thermo Scientific Pierce p-Bromophenacylate Reagent (formerly Phenacyl-8) reacts with carboxylic acids to provide quantitative detection of peptides and amino acids with few or no side reactions.

Bromophenacyl bromide is an excellent derivatization reagent for analysis of carboxylic acids. The reagent is useful for preparation of phenacyl esters, which are used to separate many saturated and unsaturated fatty acids for UV detection in HPLC applications with low nanomole sensitivity. The compound is also useful for studying acid composition of bacterial cell walls. Our p-Bromophenacylate Reagent is a ready-to-use solution of the chemical with crown ether in acetonitrile.

Features of p-Bromophenacylate Reagent:

• Ready-to-use reagent solution eliminates the need to premix of phenacylbromide and crown ether
• Derivatization is rapid and quantitative, with yields greater than 95% in 15 to 20 minutes at 80°C
• Excess reactants do not interfere with detection
• Large excess of alkylating reagent is not necessary
• Small amounts of water or alcohol do not interfere
• If isolation is desired, products are usually crystalline

Reagent Properties:

• Formulation: 0.1mmol/mL p-bromophenacyl bromide and 0.005mmol/mL crown ether in acetonitrile
• Form: Colorless to light yellow liquid
• Reactive toward: Carboxylic acids (—COOH)
• Detection: UV absorption (λmax = 260nm)
• Application: Precolumn derivatization for nanomole levels of detection by HPLC

FDAA (Marfey's Reagent) (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide) (Thermo Scientific™)

Thermo Scientific Pierce Marfey's Reagent (FDAA) reacts with primary amines to enable the quick and easy separation and quantitation of optical isomers of amino acids by reverse-phase chromatography.

Features of Marfey's Reagent (FDAA):

• For precolumn derivatization of primary amines for detection by UV absorbance (λmax = 340nm)
• Provides nanomole sensitivity in typical HPLC applications with amino acids
• Reaction products enable chiral separation of D- and L-amino acids
• Following reverse-phase HPLC, derivatives have an extinction coefficient of 3 x 10^4M-1 cm-1

FDAA is 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide, also called Marfey's reagent. The compound reacts with primary amines and is used as a derivatization reagent for UV detection in liquid chromatography methods. The reagent provides for detection at 340nm with nanomole sensitivity. FDAA derivatives of D-amino acids exhibit strong intramolecular bonding, which reduces their polarity relative to the corresponding L-amino acid derivatives. Consequently, the D-derivatives are selectively retained on reverse phase columns and elute much later than corresponding L-derivatives.

Reagent Properties:
• Alternative names: FDAA, Marfey's Reagent
• Chemical name: 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide; N2-(5-Fluoro-2,4-dinitrophenyl)-L-alaninamide
• Reactive toward: Primary amines (—NH2)
• Chemical formula: C9H9FN4O5
• CAS number: 95713-52-3
• Molecular weight: 272.19
• Form: Bright yellow or orange crystals or powder, free of particulate matter

FDAA derivatives can be separated with simple linear gradient of triethylamine phosphate/acetonitrile on a Spheri-5 RP-18 HPLC cartridge column. FDAA reduces the need for a chiral column because the D-derivatives are retained on the column, eluting after the L-derivatives. The derivatization is easy to perform and completed in 90 minutes, resulting in derivatives that are stable for at least 48 hours.

PITC (Edman's Reagent) (phenylisothiocyanate) (Thermo Scientific™)

Thermo Scientific Pierce Phenylisothiocyanate (PITC) is a high-purity reagent for pre-column quantitative derivatization of amino acids by reverse-phase HPLC.

Features of Phenylisothiocyanate (PITC):

• For pre-column derivatization of amino acids for picomole detection levels in HPLC
• Reacts with amine groups of amino acids in 5 to 10 minutes at room temperature to yield products that are detectable by UV absorbance (λmax = 254nm)
• Produces phenylthiocarbamyl (PTC) derivatives that can be separated and quantified in 30 minutes using reverse-phase HPLC
• Produces stable products with all amino acids, including proline

Phenylisothiocyanate (PITC), also known as Edman's Reagent, enables the sequential degradation of amino acids in a polypeptide chain, yielding primary structural information. PITC reacts readily with amino acids at alkaline pH. Precolumn derivatization results in phenylthiocarbamyl derivatives (PTC-amino acids) that can be separated and quantified using reverse-phase HPLC. This method produces stable products with all amino acids, including proline. PITC is volatile, making it possible to remove excess reagent in vacuo, thereby minimizing the possibility of reagent interference. Detection of picomole quantities of the derivatives can be achieved using a UV detector at 254nm. PITC derivatization followed by reverse-phase chromatography can be used for identification and quantitation of methylated, halogenated, phosphorylated and sulfonated amino acids.

Reagent Properties:
• Alternative names: PITC, Phenyl Isothiocyanate, Edman's Reagent
• Chemical name: Isothiocyanatobenzene
• Reactive toward: Primary amines (—NH2)
• Chemical formula: C7H5NS
• CAS number: 103-72-0
• Molecular weight: 135.19
• Form: Clear, colorless to pale yellow liquid

Unlike Fmoc-chloride, PITC does not yield disubstituted tyrosine or histidine derivatives. PTC-amino acids demonstrate improved stability at pH 5-7.5 as well as increased stability at room temperature over o-phthalaldehyde (OPA)-amino acid adducts. Also, unlike OPA, PITC enables the direct analysis of secondary amino acids.

TNBSA Solution (2,4,6-trinitrobenzene sulfonic acid) (5% w/v) (Thermo Scientific™)

Thermo Scientific Pierce TNBSA Solution is a 5% solution of trinitrobenzene sulfonic acid in methanol that reacts with primary amines (peptides or amino acids) to yield a soluble colored product, a property useful for various assay methods.

Features of Thermo Scientific Pierce TNBSA Solution:

• TNBSA component reacts readily with primary amino groups of amino acids in aqueous format at pH 8 to form yellow adducts
• Colored derivatives are monitored at 345nm and have extinction coefficients of 10,000 to 15,000
• Supplied as 5% solution (w/v) of TNBSA in methanol, which is easily diluted approximately 1000-fold in assay buffer for typical protein and peptide applications
• No colored derivatives are formed with secondary amino acids proline and hydroxyproline
• Applicable to solution or solid phase analysis

Properties of TNBSA:
• Alternative names: 2,4,6-trinitrobenzenesulfonic acid (TNBSA), picrylsulfonic acid, trinitrophenylsulfonic acid, trinitrobenzene sulfonate (TNBS)
• Chemical name: 2,4,6-trinitrobenzenesulfonic acid
• Reactive toward: Primary amines (—NH2)
• Chemical formula: C6H3N3O9S
• CAS number: 2508-19-2; 5400-70-4; 85600-65-3
• Molecular weight: 293.17

Example procedure for measuring protein primary amines (Hermanson, 2008, p. 127-128):
• Dissolve or dialyze protein (20-200 µg/mL) in 0.1M sodium bicarbonate buffer (pH 8.5). Avoid Tris or other amine-containing buffers.
• Dilute supplied 5% TNBSA solution 500-fold in 0.1M sodium bicarbonate buffer (pH 8.5).
• Add 0.5 mL diluted TNBSA solution to 1 mL of protein solution. Mix well.
• Incubate at 37°C for 2 hours.
• Add 0.5 mL of 10% SDS and 0.25 mL of 1N HCl to each sample to stop and stabilize reaction.
• Measure absorbance of solution at 335nm. Determine concentration of primary amines by calculation from the extinction coefficient or by comparison to amino acid standards.

This Pierce Reagent solution contains trinitrobenzene sulfonic acid (TNBSA), which reacts readily with the primary amino groups of amino acids in aqueous solution at pH 8 to form yellow adducts. No colored derivatives are formed with the secondary amino acids praline and hydroxyproline. The colored derivatives are monitored at 335 to 345nm and have extinction coefficients in the range of 10,000-15,000. TNBSA has been used as a hydrophilic modifying reagent for the detection of primary amines in samples containing amino acids, peptides or proteins. It is an excellent reagent for rapid qualitative and quantitative estimation of these biomolecules.

Pierce™ Trifluoroacetic Acid (TFA), Sequencing grade (Thermo Scientific™)

Thermo Scientific Pierce Trifluoroacetic Acid (TFA), Sequencing grade is manufactured and tested to meet strict specifications that ensure superior performance for use as an ion-pairing agent in reverse-phase peptide separations.

Features of Trifluoroacetic Acid (TFA), Sequencing grade:

High purity and exceptional clarity—allows sensitive, nondestructive peptide detection at low UV wavelengths in reverse-phase HPLC protein and peptide separation systems
High-performance packaging—TFA packaged under nitrogen in amber glass ampules or bottles with protective PTFE-lined fluorocarbon caps to ensure TFA integrity
Economical convenience—Choose the TFA format that works best for your application. In just a few seconds, 1 ml ampules can be used to prepare 1 liter of fresh 0.1% v/v trifluoroacetic acid solution for the mobile phase in reverse-phase chromatography

Applications of Trifluoroacetic Acid (TFA), Sequencing grade:
• Ion pair reagent for reverse-phase HPLC
• Protein/peptide sequencing
• Protein/peptide solubilizing agent
• Solid-phase peptide synthesis
• Amino acid analysis
• Making 0.1% solutions of trifluoroacetic acid (w/v vs. v/v)

Trifluoroacetic acid (TFA) is the most commonly used ion pairing agent for use in reverse-phase HPLC peptide separations because it sharpens peaks and improves resolution, is volatile and easily removed, has low absorption within detection wavelengths. It is manufactured to the highest specifications to ensure the integrity of your data, maximize sensitivity in your assay and to prolong the life of your equipment.

Making 0.1% Trifluoracetic Acid Solutions:
For complex peptide separations, the key to success can be to vary selectivity. Varying mobile phase composition on the same column can change selectivity enough to resolve peptides that would otherwise overlap. Trifluoroacetic acid is the most frequently used modifier for peptide separations in reverse-phase HPLC. The TFA concentration usually specified is 0.1%. For reproducible separations from run-to-run or from lab-to-lab, it is essential to make TFA concentrations the same.

Trifluoroacetic acid concentration can and should be specified as either "w/v" (weight/volume), or as "v/v" (volume/volume). The w/v specification designates that the TFA is to be weighed and added to a volume of mobile phase (e.g. 0.1% TFA w/v requires one gram of TFA per liter). The v/v specification designates that the TFA is to be measured by volume (e.g. 0.1% TFA v/v requires one mL of TFA per liter).

Because the density of trifluoroacetic acid is 1.53 g/ml the difference between 0.1% TFA (w/v) and 0.1% TFA (v/v) is more than 50%. For the sake of reproducibility, it is essential for authors of a method to specify, and for users of a method to know, whether the TFA concentration is given as "w/v" or "v/v".

Related Products
Pierce™ Trifluoroacetic Acid (TFA), LC-MS Grade