Many amino acid analysis techniques exist, and the choice of any one technique often depends on the sensitivity required from the assay. In general, about one-half of the amino acid analysis techniques employed rely on the separation of the free amino acids by ion-exchange chromatography followed by postcolumn derivatization (e.g., with ninhydrin or o-phthalaldehyde). Postcolumn detection techniques can be used with samples that contain small amounts of buffer components, such as salts and urea, and generally require between 5 and 10 µg of protein sample per analysis. The remaining amino acid techniques typically involve precolumn derivatization of the free amino acids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl carbonate; (dimethylamino)azobenzenesulfonyl chloride; 9-fluorenyl-methylchloroformate; and 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by reverse-phase HPLC. Precolumn derivatization techniques are very sensitive and usually require between 0.5 and 1.0 µg of protein sample per analysis but may be influenced by buffer salts in the samples. Precolumn derivatization techniques may also result in multiple derivatives of a given amino acid, which complicates the result interpretation. Postcolumn derivatization techniques are generally influenced less by performance variation of the assay than precolumn derivatization techniques.
Method 1Postcolumn Ninhydrin Detection
Ion-exchange chromatography with postcolumn ninhydrin detection is one of the most common methods employed for quantitative amino acid analysis. As a rule, a Li-based cation-exchange system is employed for the analysis of the more complex physiological samples, and the faster Na-based cation-exchange system is used for the more simplistic amino acid mixtures obtained with protein hydrolysates (typically containing 17 amino acid components). Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH and cation strength. A temperature gradient is often employed to enhance separation.
When the amino acid reacts with ninhydrin, the reactant has characteristic purple or yellow color. Amino acids, except imino acids, give a purple color, and show maximum absorption at 570 nm. The imino acids, such as proline, give a yellow color, and show maximum absorption at 440 nm. The postcolumn reaction between ninhydrin and amino acid eluted from the column is monitored at 440 nm and 570 nm, and the chromatogram obtained is used for the determination of amino acid composition.
The detection limit is considered to be 10 pmol for most of the amino acid derivatives, but 50 pmol for proline. Response linearity is obtained in the range of 20 to 500 pmol with correlation coefficients exceeding 0.999. To obtain good compositional data, samples larger than 1 µg before hydrolysis are best suited for this amino acid analysis of protein/peptide.
One method for postcolumn ninhydrin detection is shown below. Many other methods are also available, with instruments and reagents available commercially.
Mobile Phase Preparation
Solution A
Transfer about 1.7 g of anhydrous sodium citrate and 1.5 mL of hydrochloric acid to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Adjust, if necessary, with hydrochloric acid to a pH of 3.0.
Solution B
Transfer about 1.7 g of anhydrous sodium citrate and 0.7 mL of hydrochloric acid to a 100-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Adjust, if necessary, with hydrochloric acid to a pH of 4.3.
Solution C
Prepare a solution containing 5% of sodium chloride, 1.9% of anhydrous sodium citrate, and 0.1% of phenol in water, and adjust to a pH of 6.
Column Regeneration Solution
Prepare a solution containing 0.8% of sodium hydroxide in water, and adjust to a pH of 13.
Mobile Phase
Use variable mixtures of Solution A, Solution B, and Solution C as directed for Chromatographic System.
Postcolumn Reagent
Transfer about 18 g of ninhydrin and 0.7 g of hydrindantin to 900 mL of a solution containing 76.7% of dimethyl sulfoxide, 0.7% of dihydrate lithium acetate, and 0.1% of acetic acid, and mix for at least 3 hours under inert gas, such as nitrogen.
[noteThis reagent is stable for 30 days if kept between 2
and 8
under inert gas.
]
Buffer Solution
Prepare a solution containing 2% of anhydrous sodium citrate, 1% of hydrochloric acid, 0.5% of thiodiglycol, and 0.1% of benzoic acid in water, and adjust to a pH of 2.
Chromatographic System
The liquid chromatograph is equipped with a detector with appropriate interference filters at 440, 570, or 690 nm and a 4.0-mm × 120-mm column that contains 7.5-µm sulfonated styrene-divinylbenzene copolymer packing. The flow rate is about 14 mL per hour. The system is programmed as follows. Initially equilibrate the column with
Solution A; at 25 minutes, the composition of the
Mobile Phase is changed to 100%
Solution B; and at 37 minutes, the composition is changed to 100%
Solution C. At 75 minutes into the run, the last amino acid has been eluted from the column, and the column is regenerated with
Column Regeneration Solution for 1 minute. The column is then equilibrated with
Solution A for 11 minutes before the next injection. The column temperature is programmed as follows. The initial temperature is 48
; after 11.5 minutes, the temperature is increased to 65
at a rate of 3
per minute; at about 35 minutes, the temperature is increased to 77
at a rate of 3
per minute; and finally at about 52 minutes, the temperature is decreased to 48
at a rate of 3
per minute.
Procedure and Postcolumn Reaction
Reconstitute the lyophilized protein/peptide hydrolysate in the
Buffer Solution, inject an appropriate amount into the chromatograph, and proceed as directed for
Chromatographic System. As the amino acids are eluted from the column, they are mixed with the
Postcolumn Reagent, which is delivered at a flow rate of 7 mL per hour, through a tee. After mixing, the column effluent and the
Postcolumn Reagent pass through a tubular reactor at a temperature of 135
, where a characteristic purple or yellow color is developed. From the reactor, the liquid passes through a colorimeter with a 12-mm flow-through cuvette. The light emerging from the cuvette is split into three beams for analysis by the detector with interference filters at 440, 570, or 690 nm. The 690-nm signal may be electronically subtracted from the other signals for improved signal-to-noise ratios. The 440-nm (imino acids) and the 570-nm (amino acids) signals may be added in order to simplify data handling.
Method 2Postcolumn OPA Fluorometric Derivatization
Ion-exchange chromatography with postcolumn o-phthalaldehyde (OPA) fluorometric detection is used. The procedure employs an ion-exchange column for separation of free amino acids followed by postcolumn oxidation with sodium hypochlorite and derivatization using OPA and N-acetyl-l-cysteine. The sodium hypochlorite oxidation step allows secondary amines, such as proline, to react with the OPA reagent.
OPA reacts with primary amines in the presence of thiol compound to form highly fluorescent isoindole products. This reaction is utilized for the postcolumn derivatization in analysis of amino acids by ion-exchange chromatography. The rule of the separation is the same as Method I. Instruments and reagents for this form of amino acid analysis are available commercially. Many modifications of this method exist.
Although OPA does not react with secondary amines (imino acids, such as proline) to form fluorescent substances, the oxidation with sodium hypochlorite allows secondary amines to react with OPA. The procedure employs a strongly acidic cation-exchange column for separation of free amino acids followed by postcolumn oxidation with sodium hypochlorite and postcolumn derivatization using OPA and thiol compound, such as N-acetyl-l-cysteine and 2-mercaptoethanol. The derivatization of primary amino acids are not noticeably affected by the continuous supply of sodium hypochlorite.
Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes of pH and cation strength. After postcolumn derivatization of eluted amino acids with OPA, the reactant passes through the fluorometric detector. Fluorescence intensity of OPA-derivatized amino acids are monitored with an excitation wavelength of 348 nm and an emission wavelength of 450 nm.
The detection limit is considered to be a few tens of pmol level for most of the amino acid derivatives. Response linearity is obtained in the range of a few pmol level to a few tens of nmol level. To obtain good compositional data, a sample greater than 500 ng before hydrolysis is best suited for the amino acid analysis of protein/peptide.
One method of postcolumn OPA fluorometric detection is shown below.
Mobile Phase Preparation
Solution A
Prepare a solution of sodium hydroxide, citric acid, and alcohol in HPLC grade water having a 0.2 N sodium concentration and containing 7% of alcohol (w/v), adjusted to a pH of 3.2.
Solution B
Prepare a solution of sodium hydroxide and citric acid in HPLC grade water having a 0.6 N sodium concentration, adjusted to a pH of 10.0.
Solution C:
0.2 N sodium hydroxide.
Mobile Phase
Use variable mixtures of Solution A, Solution B, and Solution C as directed for Chromatographic System.
Postcolumn Reagent Preparation
Alkaline Buffer
Prepare a solution containing 384 mM sodium carbonate, 216 mM boric acid, and 108 mM potassium sulfate, and adjust to a pH of 10.0.
Hypochlorite Reagent
To 1 L of Alkaline Buffer, add 0.4 mL of sodium hypochlorite solution (10% chlorine concentration). [noteThe hypochlorite solution is stable for 2 weeks.]
OPA Reagent
Transfer 2 g of N-acetyl-l-cysteine and 1.6 g of OPA to a 15-mL volumetric flask, dissolve in and dilute with alcohol to volume, and mix. Transfer this solution and 4 mL of 10% aqueous polyethylene (23) lauryl ether to a 1-L volumetric flask, dilute with 980 mL of Alkaline Buffer, and mix.
Chromatographic System
The liquid chromatograph is equipped with a fluorometric detector set to an excitation wavelength of 348 nm and an emission wavelength of 450 nm and a 4.0-mm × 150-mm column that contains 7.5-µm packing L17. The flow rate is about 0.3 mL per minute, and the column temperature is set at 50
. The system is programmed as follows. The column is equilibrated with
Solution A; over the next 20 minutes, the composition of the
Mobile Phase is changed linearly to 85%
Solution A and 15%
Solution B; then there is a step change to 40%
Solution A and 60%
Solution B; over the next 18 minutes, the composition is changed linearly to 100%
Solution B and held for 7 minutes; then there is a step change to 100%
Solution C, and this is held for 6 minutes; then there is a step change to
Solution A, and this composition is maintained for the next 8 minutes.
Procedure and Postcolumn Reaction
Inject about 1.0 nmol of each amino acid under test into the chromatograph, and proceed as directed for
Chromatographic System. As the effluent leaves the column, it is mixed with the
Hypochlorite Reagent. The mixture passes through the first postcolumn reactor which consists of stainless steel 0.5-mm × 2-m tubing. A second postcolumn reactor of similar design is placed immediately downstream from the first postcolumn reactor and is used for the OPA postcolumn reaction. The flow rates for both the
Hypochlorite Reagent and the
OPA Reagent are 0.2 mL per minute, resulting in a total flow rate (i.e.,
Hypochlorite Reagent,
OPA Reagent, and column effluent) of 0.7 mL per minute exiting from the postcolumn reactors. Postcolumn reactions are conducted at 55
. This results in a residence time of about 33 seconds in the OPA postcolumn reactor. After postcolumn derivatization, the column effluent passes through the fluorometric detector.
Method 3Precolumn Determination
Precolumn derivatization of amino acids with phenylisothiocyanate (PITC) followed by reverse-phase HPLC separation with UV detection is used.
PITC reacts with amino acids to form phenylthiocarbamyl (PTC) derivatives, which can be detected with high sensitivity at 254 nm. Therefore, precolumn derivatization of amino acids with PITC followed by a reverse-phase HPLC separation with UV detection is used to analyze the amino acid composition.
After the reagent is removed under vacuum, the derivatized amino acids can be stored dry and frozen for several weeks with no significant degradation. If the solution for injection is kept cold, no noticeable loss in chromatographic response occurs after three days.
Separation of the PTC-amino acids on a reverse-phase HPLC with ODS column is accomplished through a combination of changes in concentrations of acetonitrile and buffer ionic strength. PTC-amino acids eluted from the column are monitored at 254 nm.
The detection limit is considered to be 1 pmol for most of the amino acid derivatives. Response linearity is obtained in the range of 20 to 500 pmol with correlation coefficients exceeding 0.999. To obtain good compositional data, a sample larger than 500 ng of protein/peptide before hydrolysis is best suited for this amino analysis of proteins/peptides.
One method of precolumn PITC derivatization is described below.
Mobile Phase Preparation
Solution A:
0.05 M ammonium acetate, adjusted with phosphoric acid to a pH of 6.8.
Solution B
Prepare 0.1 M ammonium acetate, adjust with phosphoric acid to a pH of 6.8, and then prepare a mixture of this solution and acetonitrile (1:1).
Solution C:
a mixture of acetonitrile and water (70:30).
Mobile Phase
Use variable mixtures of Solution A, Solution B, and Solution C as directed for Chromatographic System.
Derivatization Reagent Preparation
Coupling Buffer:
a mixture of acetonitrile, pyridine, triethylamine, and water (10:5:2:3).
Sample Solvent:
a mixture of water and acetonitrile (7:2).
Sample Derivatization Procedure
Dissolve the lyophilized test sample in 100 µL of the Coupling Buffer, and then dry in a vacuum centrifuge to remove any hydrochloride if a protein hydrolysis step was used. Dissolve the test sample in 100 µL of Coupling Buffer, add 5 µL of PITC, and incubate at room temperature for 5 minutes. The test sample is again dried in a vacuum centrifuge, and is dissolved in 250 µL of Sample Solvent.
Chromatographic System
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 250-mm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 52
. The system is programmed as follows. The column is equilibrated with
Solution A; over the next 15 minutes, the composition of the
Mobile Phase is changed linearly to 85%
Solution A and 15%
Solution B; over the next 15 minutes, the composition is changed linearly to 50%
Solution A and 50%
Solution B; then there is a step change to 100%
Solution C, and this is held for 10 minutes; then there is a step change to 100%
Solution A, and the column is allowed to equilibrate before the next injection.
Procedure
Inject about 1.0 nmol of each PITC-amino acid under test (10-µL sample in Sample Solvent) into the chromatograph, and proceed as directed for Chromatographic System.
Method 4Precolumn AQC Derivatization
Precolumn derivatization of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed by reverse-phase HPLC separation with fluorometric detection is used.
AQC reacts with amino acids to form stable, fluorescent unsymmetric urea derivatives (AQC-amino acids) which are readily amenable to analysis by reverse-phase HPLC. Therefore, precolumn derivatization of amino acids with AQC followed by reverse-phase HPLC separation is used to analyze the amino acid composition.
Separation of the AQC-amino acids on an ODS column is accomplished through a combination of changes in the concentrations of acetonitrile and salt. Selective fluorescence detection of the derivatives with an excitation wavelength at 250 nm and an emission wavelength at 395 nm allows for the direct injection of the reaction mixture with no significant interference from the only major fluorescent reagent byproduct, 6-aminoquinoline. Excess reagent is rapidly hydrolyzed (t1/2< 15 seconds) to yield 6-aminoquinoline-N-hydroxysuccinimide and carbon dioxide, and after 1 minute no further derivatization can take place.
Peak areas for AQC-amino acids are essentially unchanged for at least 1 week at room temperature, and the derivatives have more than sufficient stability to allow for overnight automated chromatographic analysis.
The detection limit is considered to be ranging from about 40 fmol to 320 fmol for each amino acid, except for Cys. The detection limit for Cys is approximately 800 fmol. Response linearity is obtained in the range of 2.5 µM to 200 µM with correlation coefficients exceeding 0.999. Good compositional data can be obtained from the analysis of derivatized protein hydrolysates containing as little as 30 ng of protein/peptide.
One method of precolumn AQC derivatization is shown below.
Mobile Phase Preparation
Solution A
Prepare a solution having a composition of 140 mM sodium acetate and 17 mM triethylamine, and adjust with phosphoric acid to a pH of 5.02.
Solution B:
a mixture of acetonitrile and water (60:40).
Mobile Phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic System.
Sample Derivatization Procedure
Dissolve about 2 µg of the test sample in 20 µL of 15 mM hydrochloric acid, and dilute with 0.2 M borate buffer (pH 8.8) to 80 µL. The derivatization is initiated by the addition of 20 µL of 10 mM AQC in acetonitrile, and allowed to proceed for 10 minutes at room temperature.
Chromatographic System
The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 250 nm and an emission wavelength of 395 nm and a 3.9-mm × 150-mm column that contains 4-µm packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 37
. The system is programmed as follows. The column is equilibrated with
Solution A; over the next 0.5 minute, the composition of the
Mobile Phase is changed linearly to 98%
Solution A and 2%
Solution B; then over the next 14.5 minutes to 93%
Solution A and 7%
Solution B; then over the next 4 minutes to 87%
Solution A and 13%
Solution B; over the next 14 minutes to 68%
Solution A and 32%
Solution B; then there is a step change to 100%
Solution B for a 5-minute wash; over the next 10 minutes, there is a step change to 100%
Solution A; and the column is allowed to equilibrate before the next injection.
Procedure
Inject about 0.05 nmol of each AQC-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.
Method 5Precolumn OPA Derivatization
Precolumn derivatization of amino acids with OPA followed by reverse-phase HPLC separation with fluorometric detection is used. This technique does not detect amino acids that exist as secondary amines (e.g., proline).
OPA in conjunction with a thiol reagent reacts with primary amine groups to form highly fluorescent isoindole products. 2-Mercaptoethanol and 3-mercaptopropionic acid can be used as thiol. OPA itself does not fluoresce and consequently produces no interfering peaks. In addition, its solubility and stability in aqueous solution, along with the rapid kinetics for the reactions, make it amenable to automated derivatization and analysis using an autosampler to mix the sample with the reagent. However, lack of reactivity with secondary amino acids has been a predominant drawback. This method does not detect amino acids that exist as secondary amines (e.g., proline). To compensate for this drawback, this technique may be combined with another technique described in Method 7 or Method 8.
Precolumn derivatization of amino acids with OPA is followed by reverse-phase HPLC separation. Because of the instability of the OPA-amino acid derivative, HPLC separation and analysis are performed immediately following derivatization. The liquid chromatograph is equipped with a fluorometric detector for the detection of derivatized amino acids. Fluorescence intensity of the OPA-derivatized amino acids are monitored with an excitation wavelength of 348 nm and an emission wavelength of 450 nm.
The detection limits as low as 50 fmol via fluorescence have been reported, although the practical limit of analysis remains at 1 pmol. One method of precolumn OPA derivatization is shown below.
Mobile Phase Preparation
Solution A:
a mixture of 100 mM sodium acetate (pH 7.2), methanol, and tetrahydrofuran (900:95:5).
Solution B:
methanol.
Mobile Phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic System.
Derivatization Reagent
Dissolve 50 mg of OPA in 1.25 mL of methanol (protein sequencing grade). Add 50 µL of 2-mercaptoethanol and 11.2 mL of 0.4 M sodium borate (pH 9.5), and mix. [noteThis reagent is stable for 1 week.]
Sample Derivatization Procedure
Transfer about 5 µL of the test sample to an appropriate container, add 5 µL of the Derivatization Reagent, and mix. After 1 minute, add not less than 20 µL of 0.1 M sodium acetate (pH 7.0). Use 20 µL of this solution for analysis. [noteUse of an internal standard (e.g., norleucine) is recommended for quantitative analysis because of potential reagent volume variations in the sample derivatization. The sample derivatization is performed in an automated on-line fashion. Because of the instability of the OPA-amino acid derivative, HPLC separation and analysis are performed immediately following derivatization.]
Chromatographic System
The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 348 nm and an emission wavelength of 450 nm and a 4.6-mm × 75-mm column that contains 3-µm packing L3. The flow rate is about 1.7 mL per minute, and the column temperature is maintained at 37
. The system is programmed as follows. The column is equilibrated with 92%
Solution A and 8%
Solution B; over the next 2 minutes, the composition of the
Mobile Phase is changed to 83%
Solution A and 17%
Solution B, and held for an additional 3 minutes; then changed to 54%
Solution A and 46%
Solution B over the next 5 minutes, and held for an additional 2 minutes; then changed to 34%
Solution A and 66%
Solution B over the next 2 minutes, and held for 1 minute; then over the next 0.3 minute changed to 20%
Solution A and 80%
Solution B, and held for an additional 2.6 minutes; and then finally over 0.6 minute changed to 92%
Solution A and 8%
Solution B, and held for an additional 0.6 minute.
Procedure
Inject about 0.02 nmol of each OPA-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.
Method 6Postcolumn DABS-Cl Derivatization
Precolumn derivatization of amino acids with (dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) followed by reverse-phase HPLC separation with visible light detection is used.
DABS-Cl is a chromophoric reagent employed for the labeling of amino acids. Amino acids labeled with DABS-Cl (DABS-amino acids) are highly stable and show the maximum absorption at 436 nm.
DABS-amino acids, all 19 naturally occurring amino acids derivatives, can be separated on an ODS column of a reverse-phase HPLC by employing gradient systems consisting of acetonitrile and aqueous buffer mixture. Separated DABS-amino acids eluted from the column are detected at 436 nm in the visible region.
This method can analyze the imino acids, such as proline, together with the amino acids, at the same degree of sensitivity. DABS-Cl derivatization method permits the simultaneous quantification of tryptophan residues by previous hydrolysis of the protein/peptide with sulfonic acids, such as mercaptoethanesulfonic acid, p-toluenesulfonic acid, or methanesulfonic acid, described for Method 2 in Protein Hydrolysis under Amino Acid Analysis. The other acid-labile residues, asparagine and glutamine, can also be analyzed by previous conversion into diaminopropionic acid and diaminobutyric acid, respectively, by treatment of protein/peptide with BTI, described for Method 11 in Protein Hydrolysis under Amino Acid Analysis.
The nonproteinogenic amino acid, norleucine, cannot be used as an internal standard in this method as this compound is eluted in a chromatographic region crowded with peaks of primary amino acids. Nitrotyrosine can be used as an internal standard because it is eluted in a clean region.
The detection limit of DABS-amino acid is about 1 pmol. As little as 2 to 5 pmol of an individual DABS-amino acid can be quantitatively analyzed with reliability, and only 10 ng to 30 ng of the dabsylated protein hydrolysate is required for each analysis.
One method for precolumn DABS-Cl derivatization is shown below.
Mobile Phase Preparation
Solution A:
25 mM sodium acetate (pH 6.5) containing 4% of dimethylformamide.
Solution B:
acetonitrile.
Mobile Phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic System.
Derivatization Reagent Preparation
Sample Buffer:
50 mM sodium bicarbonate, adjusted to a pH of 8.1.
Derivatization Reagent
Dissolve 1.3 mg of DABS-Cl in 1 mL of acetonitrile. [noteThis reagent is prepared fresh shortly before the derivatization step.]
Sample Dilution Buffer
Prepare a mixture of 50 mM sodium phosphate (pH 7.0) and alcohol (1:1).
Sample Derivatization Procedure
Dissolve the test sample in 20 µL of
Sample Buffer, add 40 µL of
Derivatization Reagent, and mix. The sample container is sealed with a silicon-rubber stopper, and heated to 70
for 10 minutes. During the sample heating, the mixture will become completely soluble. After the derivatization, dilute the test sample with an appropriate quantity of the
Sample Dilution Buffer.
Chromatographic System
The liquid chromatograph is equipped with a 436-nm detector and a 4.6-mm × 250-mm column that contains packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 40
. The system is programmed as follows. The column is equilibrated with 85%
Solution A and 15%
Solution B; over the next 20 minutes, the composition of the
Mobile Phase is changed to 60%
Solution A and 40%
Solution B; over the next 12 minutes, the composition is changed to 30%
Solution A and 70%
Solution B, and held for an additional 2 minutes.
Procedure
Inject about 0.05 nmol of the DABS-amino acids into the chromatograph, and proceed as directed for Chromatographic System.
Method 7Precolumn FMOC-Cl Derivatization
Precolumn derivatization of amino acids with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC separation with fluorometric detection is used.
FMOC-Cl reacts with both primary and secondary amino acids to form highly fluorescent products. The reaction of FMOC-Cl with amino acid proceeds under mild conditions, in aqueous solution, and is completed in 30 seconds. The derivatives are stable, with only the histidine derivative showing any breakdown. Although FMOC-Cl is fluorescent itself, the reagent excess and fluorescent side-products can be eliminated without loss of FMOC-amino acids.
FMOC-amino acids are separated by reverse-phase HPLC using an ODS column. The separation is carried out by gradient elution varied linearly from a mixture of acetic acid buffer, methanol, and acetonitrile (50:40:10) to a mixture of acetonitrile and acetic acid buffer (50:50), and 20 amino acid derivatives that are separated in 20 minutes. Each derivative eluted from the column is monitored by a fluorometric detector set at an excitation wavelength of 260 nm and an emission wavelength of 313 nm.
The detection limit is in the low fmol range. A linearity range of 0.1 µM to 50 µM is obtained for most amino acids.
One method for precolumn FMOC-Cl derivatization is shown below.
Mobile Phase Preparation
Acetic Acid Buffer
Transfer 3 mL of glacial acetic acid and 1 mL of triethylamine to a 1-L volumetric flask, and dilute with HPLC grade water to volume. Adjust with sodium hydroxide to a pH of 4.20.
Solution A:
a mixture of Acetic Acid Buffer, methanol, and acetonitrile (50:40:10).
Solution B:
a mixture of acetonitrile and Acetic Acid Buffer (50:50).
Mobile Phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic System.
Derivatization Reagent Preparation
Borate Buffer
Prepare a 1 M boric acid solution, and adjust with sodium hydroxide to a pH of 6.2.
FMOC-Cl Reagent
Dissolve 155 mg of 9-fluorenylmethyl chloroformate in 40 mL of acetone, and mix.
Sample Derivatization Procedure
To 0.4 mL of the test sample add 0.1 mL of Borate Buffer and 0.5 mL of FMOC-Cl Reagent. After about 40 seconds, extract the mixture with 2 mL of pentane, and then extract again with fresh pentane. The aqueous solution with amino acid derivatives is then ready for injection.
Chromatographic System
The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 260 nm and an emission wavelength of 313 nm and a 4.6-mm × 125-mm column that contains 3-µm packing L1. The flow rate is about 1.3 mL per minute. The system is programmed as follows. The column is equilibrated with Solution A, and this composition is maintained for 3 minutes; over the next 9 minutes, it is changed to 100% Solution B; then over the next 0.5 minute, the flow rate is increased to 2 mL per minute, and held until the final FMOC-amino acid is eluted from the column. The total run time is about 20 minutes.
Procedure
Inject not less than 0.01 nmol of each FMOC-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System. The FMOC-histidine derivative will generally give a lower response than the other derivatives.
Method 8Precolumn NBD-F Derivatization
Precolumn derivatization of amino acids with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) followed by reverse-phase HPLC separation with fluorometric detection is used.
7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reacts with both primary and secondary amino acids to form highly fluorescent products. Amino acids are derivatized with NBD-F by heating to 60
for 5 minutes.
NBD-amino acid derivatives are separated on an ODS column of reverse-phase HPLC by employing a gradient elution system consisting of acetonitrile and aqueous buffer mixture, and 17 amino acid derivatives that are separated in 35 minutes. E-aminocaproic acid can be used as an internal standard because it is eluted in a clean chromatographic region. Each derivative eluted from the column is monitored by a fluorometric detector set at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.
The sensitivity of this method is almost the same as that for the precolumn OPA derivatization method (Method 5), excluding proline to which OPA is not reactive and might be advantageous for NBD-F against OPA.
The detection limit for each amino acid is about 10 fmol. Profile analysis was achieved for about 1.5 mg of protein hydrolysates in the final precolumn labeling reaction mixture for HPLC.
One method for precolumn NBD-F derivatization is shown below.
Mobile Phase Preparation
Solution A:
a solution of 10mM sodium citrate containing 75 mM sodium perchlorate, adjusted with hydrochloric acid to a pH of 6.2.
Solution B:
a mixture of acetonitrile and water (50:50).
Derivatization Reagent Preparation
Sample Buffer:
a 0.1 M boric acid solution, adjusted with sodium hydroxide to a pH of 9.2.
Derivatization Reagent
Dissolve 5 mg of NBD-F in 1.0 mL of alcohol, and mix.
Sample Derivatization Procedure
Dissolve the test sample in 20 µL of
Sample buffer, add 10 µL of
Derivatization Reagent, and mix. The sample container is heated at 60
for 5 minutes. After the derivatization, dilute the test sample with 300 µL of
Solution A.
Chromatographic System
The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 480 nm and an emission wavelength of 530 nm and a 4.6-mm × 150-mm column that contains 5-µm particle size ODS silica packing. The flow rate is about 1.0 mL per minute, and the column temperature is maintained at 40
. The system is programmed as follows. The column is equilibrated with 94%
Solution A and 6%
Solution B; over the next 16 minutes, the composition is changed linearly to 63%
Solution A and 37%
Solution B; over the next 5 minutes, the composition is changed linearly to 62%
Solution A and 38%
Solution B; over the next 9 minutes, the composition is changed linearly to 100%
Solution B, and held for an additional 5 minutes; then finally over 2 minutes, the composition is changed linearly to 94%
Solution A and 6%
Solution B; and then the column is allowed to equilibrate before the next injection.
Procedure
Inject about 15 pmol of each NBD-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.