2. Methods of analysis of amino acids in foods.
3. Amino acid separation techniques
4. Derivatization reagents.
5. Pre-column derivatization versus post-column
Amino acids, peptides and proteins are important constituents of food. On the one hand provide the elements necessary for protein synthesis. Furthermore, amino acids and peptides contribute directly to the flavor of foods and are precursors of aromatics and colored substances formed by the thermal reaction and / or enzyme occurring during procurement, preparation and storage thereof.
Amino acid. Amino acids are carboxylic acids containing an amino group. There are about a hundred in nature. In total protein hydrolyzate there are twenty amino acids, with some exceptions, the following general formula:
In the simplest case, the radical R is a hydrogen atom (aminoacetic acid or glycine), other amino acids in R is an aliphatic, aromatic or heterocyclic group, which may carry other functional groups.
There are ten amino acids, called essential amino acids that the body can not synthesize and must be supplied by food. The body can not synthesize it lacks the necessary machinery to produce the corresponding keto acids, it has been found that if you provide them and ammonium salts can make them.
The constituent amino acids of proteins are approximately twenty: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
They are but two amino acids, which are imino acids, proline and hydroxyproline, which amino acids may also be considered by their structural similarity. They are called amino acids because the amino group is attached to the carbon chain is, by convention, the carbon atom adjacent to the carboxyl group.
Amino acids containing many acidic and basic groups, therefore have weak acidic and basic properties and are called ampholytes. Amphiprotic behave as they may accept or donate electrons. Amino acid molecules carry a negative and a positive charge, which results in a null total load, this form is called “zwitterion” amino acid. The total charge transported by an amino acid depends on the pH of the solution and the values of the acidity constant of ionizable groups present. If the pH is greater than the constant value of a group, a proton is released and the molecule will have a negative charge, but if the pH is lower, will have a positive charge. The fact that different pH values to the amino take various forms and have different net charges are used in many analytical methods, such as electrophoresis and ion exchange chromatography. The iso-ionic point of a molecule is the pH at which the number of negative charges due to protons lost equals the number of positive charges due to protons cattle, then zwitterionic form predominates. The isoelectric point is pH at which the molecules do not present migration in an electric field and can be determined experimentally by electrophoresis; at amino acids is equal to the iso-ionic point.
An important aspect in the analysis of amino acids that is not detectable in the visible-UV. There are several reagents which react with amino acids resulting colored or fluorescent compounds and therefore can be used for qualitative or quantitative analysis. This is what is called the derivatization of amino acids. Fluorimetric methods have many advantages over spectrophotometry for amino acid analysis.
Protein needs. It requires a criterion for setting the needs or requirements for the human protein. Thus establishing a “reference protein” or “pattern”.
Known amino acid composition of proteins of different foods consumed and which have been shown to possess greater biological value, namely that of milk, egg and meat, and taking into account the qualitative and quantitative composition amino acid, has adopted this “reference protein” or “pattern”.
The quality of proteins influencing the content of essential amino acids, and it is important the presence of “limiting”. In effect, the so called essential amino acid in a protein is given in lower relative amounts with respect to protein pattern because it limits the use of this protein to the biological purposes.
It is important to determine the amino acid content, together, to estimate the degree of enzymatic proteolysis which has undergone the product and the proportion of each of them as components of the protein when it is desired to establish their biological value.
The determination of amino acids in foods some conclusions about their composition, for example, gelatin is used as thickener quick and simple difference, other hydrocolloids (vegetable fats, starch, etc.), Depending on their amino acid composition.
First collect the various official methods and standards for the analysis of amino acids in food, found in Official Methods of Analysis published by the Ministry of Agriculture, Fisheries and Food (1986) in Methods of Analysis of AOAC (1995) and some recommended methods.
Later in the discussion, we discussed the various separation techniques (chromatography and electrophoresis), automatic amino acid analyzer, of different derivatizing reagents and detection type appropriate for each, and the advantages and disadvantages of derivatization, both before (pre-column) and after (post-column) of the separation.
2. Methods of analysis of amino acids in foods.
Determination of proline in honey.
Proline reaction with ninhydrin in acidic medium and subsequent determination at 517 nm of the absorbance of the compound formed.
Sample preparation: Assuming that honey has been removed from the panel by runoff through a mesh filter, we will choose to make the next step, homogenization. Softening by heating the sample at 25-30 C while stirring. Once homogenized heated to 50 C water bath until fusion is achieved quickly and allowed to cool.
Determination of hydroxyproline in meat.
Upon acidic hydrolysis of the protein and oxidation of hydroxyproline. The derivative formed with p-dimethylaminobenzaldehyde is measured colorimetrically at 560 nm ..
Sample preparation: First, remove the protective layers of the product to take a representative sample. Are broken pieces of 0.5 to 1 cm and said pieces are cut in small cubes. After passed through a crusher several times until obtaining a homogenous mixture. Vials were stored in clean and dry to prevent loss of moisture. Kept refrigerated so that prevents deterioration and any change in its composition.
Determination of proline in honey.
Determination of hydroxyproline in meat.
Determination of amino acid vitamin preparations.
Determination of lysine in nutritional supplements.
The amino groups of proteins react with DNFB resulting DNFB lysine derivatives. Subsequently acidic hydrolysis occurs and the determination of an amino acid analyzer lysine.
Determination of tryptophan in foods.
The sample is hydrolysed in basic medium, followed by a pH adjustment made. Tryptophan is separated by ion exchange chromatography and liquid determined by a UV detector at 280 nm.
Determination of sulfur amino acids in foods (methionine and cysteine).
First the proteins are oxidized with performic acid and cysteic acid to obtain sulfonated methionine, then acid hydrolysis is performed with HCl. We ion exchange chromatography by applying a detector that is capable of measuring at 570 nm.
Amino Acid Analysis by HPLC followed phenylisothiocyanate method.
(Lab. Enzymology and Physical Chemistry of Proteins).
Next, a typical protocol included the analysis of amino acids in flour:
– Allow the sample to reflux with 6N HCl for 24 hours. Evaporate to dryness in rotary evaporator.
– Add water and evaporate to dryness.
– Perform a chromatographic test sample solution 10% in the treated sample solution – aqueous isopropanol at 10%.
– The solvent system: n-butanol/Hac/H2O in 12:3:5 ratio.
– Whatman paper No 1.
– Paper length 50 cm.
– Descending chromatographic method.
– Duration: 12 hours.
– Developing solution: ninhydrin acetone solution 0.25%.
– Developing conditions: 20 min at 105 C.
– Comparison of Pure samples versus amino acid hydrochlorides.
3. Amino acid separation techniques.
It is often necessary to identify and quantify the individual amino acids in a mixture, both metabolic studies as the research on protein structure.
The use of thin layer chromatography or electrophoresis are suitable to determine the number and relative amount of the various amino acids present in a sample (qualitative analysis), but for the quantitative analysis is necessary or gas chromatography analyzer amino acids.
Thin layer chromatography.
An important application of thin layer chromatography is to serve as a guide for developing optimal conditions for liquid separations by column chromatography. Provides a quick overview of the major amino acid present in the sample. The advantages of this method are speed and low cost of the experiments. In fact, some chromatographers are of the opinion that the thin layer tests should always precede the use of the column
In the paper chromatography can apply large sample volumes, allowing subsequent elution of a particular amino acid for subsequent purification and analysis, it is very important factor in the identification of an unknown sample constituent.
Before performing the chromatography, it is necessary to remove interfering substances such as proteins, carbohydrates
and salts, which can be done with ion exchange resin. Amino acids eluted retained can then adding the column to a small volume of ammonia and then washing with distilled water.
The separation of amino acids is based in that they are differentially distributed between the mobile phase and the stationary phase. Generally the procedure is carried upward by: inserting the lower edge of the plate in the chromatographic eluent action to be sucked by capillary forces of the surface coating of the plate.
The identification of an amino acid is performed by comparing the Rf values (delay factor: ratio between the distance traveled by the compound from the origin and the distance traveled by the solvent from the origin), other taken as reference, it being necessary to use at least three different solvent systems to establish their identity with a certain degree of security.
The nature of amino acids is an important factor when choosing a solvent, because different solvents may enable better resolution components of acidic, basic or neutral. In general, increasing the proportion of water in the solvent increases all values of Rf and introducing small amounts of ammonia increase the delay factor values of the basic amino acids. Some solvents contain harmful compounds, such as phenol, which restricts its routine use. The chemical composition of the solvent can also limit the range of reagents that can be applied satisfactorily location. For example, sulfanilic acid can not be used with phenolic solvents.
The resolving power of the thin layer chromatography using two-dimensional techniques can be increased, that is, using two different solvents. Applying a sample quantity greater than the one usually used for the chromatographic separation of a dimension (about three times) in a corner of the paper or board and is run in one dimension. The chromatogram then thoroughly air dried, rotated an angle of 90 and is developed with the second solvent. After further drying, is immersed in the reagent chosen location. In the two-dimensional chromatography, the solvent composition of the two that determines the order in which to use.
Two-dimensional separations allow resolution of a large number of amino acids present in a sample, since those with a mobility similar to the first dimension separates the second. This is very useful in detecting the components that are very low in concentration and may be masked by other compounds having a high concentration when using chromatography in one dimension.
Electrophoresis is a process in which charged species (ions or colloidal particles) are separated according to their different rates of migration in an electric field. Since the Fifties, electrophoretic separations were the cornerstone of much research on chemical and molecular biologists related to the separation and analysis of proteins, polynucleotides and other biopolymers. These separations have been (and still are) very efficient and extensive application, but unfortunately, they are a very slow and laborious techniques that tend to be poorly reproducible.
In the mid-eighties, this situation changed dramatically with the emergence of commercial devices for microscale analytical electrophoresis in capillary columns (capillary electrophoresis).
The fact that the various amino acids carrying different net charges at a particular pH, can be separated from a mixture by electrophoresis of high or low voltage. The most common means used as support are paper or thin-layer plates (cellulose or silica gel) and can be used to visualize the spots, the location reagents described later. Separations can be performed more easily with low voltage high, one of the main advantages of the first, salts and other substances present in the sample lesser extent affect the quality of the electrophoretogram.
Although electrophoretic separations can be achieved with buffers at various pH, in practice chosen pH values are between 2 and 5’3. At pH 2 all amino acids are positively charged and the basic type with the highest positive charge migrating more rapidly toward the cathode, while that at pH 5 amino acids are directed toward both electrodes, depending on the load carried. 5’3 pH separations are used to determine the nature of an acidic or basic amino unknown.
There have been many attempts to harness the speed and sensitivity offered by gas chromatography, but although there has been considerable progress in the development of such methods are not yet routinely used for the analysis of amino acids in biological samples. The reason for this lies in the fact that amino acids, although similar compounds are also chemically heterogeneous and are not sufficiently volatile to become less than some appropriate derivative. Difficulties arise not only when choosing the method for obtaining such derivatives, but also in the selection of a stationary phase that is capable of resolving all elements derived from a diverse group of compounds. In the choice of the detector are also similar problems.
Automatic amino acid analyzer – HPLC.
There are two techniques for the determination of amino acids by liquid chromatography: partition chromatography in reverse phase and ion exchange chromatography.
The high performance liquid chromatography resolution separation technique is the most widely used. The most important reasons are its sensitivity, its easy adaptation to accurate quantitative determinations, their suitability for the separation of non-volatile or thermolabile and, above all, its great applicability to substances that are of primary interest in the industry in many fields of science and society in general. Examples of such materials include amino acids, proteins, nucleic acids and others.
Partition chromatography on reversed phase.
Reverse phase chromatography is a predominantly polar solvent as mobile phase and a hydrocarbon chain linked as stationary phase. Some of the most significant advantages of this approach are its high reproducibility, short retention times, high sample rate, single chromatographic system and wide applicability. Therefore, applications are becoming increasingly numerous.
Selectivity is based on specific interactions between the solute and the mobile phase, either polar type, forming hydrogen bonds or by secondary equilibria caused to vary the composition of the mobile phase (acid-base complex formation or ion pairs, addition of metal-ligand complexes or chiral reagents to separate optical isomers, etc.).
Ion exchange chromatography.
Ion chromatography is related to modern and efficient methods for the determination of ions based on the use of ion exchange resins.
There automated methods for separation and detection of amino acids and other ionic species in complex mixtures. The development of modern HPLC began in the late sixties, but its application to ion exchange separation was delayed due to the lack of a general and sensitive method for the detection of species such as alkali and alkaline earth cations and anions as halides, nitrate and acetate. This situation was remedied in 1975 to develop by Dow Chemical Company Technical suppression technique effluent, which makes possible the detection of ions eluted conductivity. The amino acid analyzer, with which it performs the quantization of the amino acids is based on this technique.
Automatic amino acid analyzer. The amino acid separation process was first automated chromatographic post-column derivatization. The separation was carried out by Moore, Spackman and Stein (1958), by ion exchange, followed by quantization of each component eluted from the column ninhydrin reaction and subsequent detection in the visible. This method has been performed for more than thirty years, in any laboratory of proteins.
The equipment currently used is based on the original while introducing some modifications for better efficiency. Involves pumping pH buffers or ionic strength varying through a thermostated column. Among the new resins is the production of high quality development HPLC sophisticated automation systems and increased sensitivity of detection methods.
The separation takes place in a column of sulfonated polystyrene resin. Initially, the pH of the mobile phase is low, so that the positively charged amino acids will be attracted to sulfur groups (SO3-). As the pH is increased amino acid buffer differentially eluted by decreasing its positive charge and be less attracted by the anion. At pH 3.5 the amino acid group with an extra, your chain will have very little affinity for the resin and will be the first out of the column, while having an extra set ionizable capable of carrying a positive charge (eg lysine, histidine) will be strongly retained by the resin and will be eluted only when the pH has been increased substantially and reduced net positive charge.
Besides the pH must also consider the concentration of eluting buffer, elution by influencing such that when the ion concentration increases in the same, amino acids are eluted faster.
The ionic interactions between the resin and the amino acids also influence the sequence of elution, allowing some amino acids having a similar behavior elute separately.
The quantitative resolution of mixtures of amino acids is achieved by varying the buffer composition, by increasing the pH and maintaining a constant concentration, or vice versa, or even by varying both. Other less significant factor in the quality of separation such as temperature, flow rate of buffer or the type of cation used.
Practical aspects of the analyzer:
Column: type analyzers using two columns, one to separate the acidic and neutral amino acids and one for the basics. The glass or stainless steel and tall narrow peaks occur, improving the separation of similar amino acid.
Buffer solution: it is usually lithium or sodium citrate, which is added a detergent, an antioxidant and a preservative. Currently, two buffers are used, one acidic and one basic. Are mixed in varying proportions to achieve an increase in pH. This separation is improved by reducing the analysis time and eliminates the sharp fluctuations of the baseline due to sudden changes in the buffer, as with previous techniques.
Temperature: can be constant to avoid changes in pH and the ionization of amino acids, or you can use a temperature program that involves changing it in various stages of procedure.
Column flow: it requires a constant flow of buffer to allow reproducibility. Is achieved by use of pressure pumps or constant displacement.
Detection: second pump is needed for the derivatizing reagent (usually ninhydrin) to be mixed with the effluent from the column. When the reaction has taken place, the current is controlled in a flow cell of a colorimeter or fluorimeter. Continuously measuring the absorbance at 570 and 440 nm. to detect the amino and imino respectively.
The amino acid analysis is a good example of the fact that there is rarely a single separation system for analytical problems, but the choice of the appropriate system depends on the analytical problem. For example, the matrix in which the amino acids will be determined by the desired sensitivity and speed required for analysis.
Current methods for amino acid detection rely on the reaction of primary amino group of the amino acid to form a colored or fluorescent derivative, this derivatization occurs both before and after the separation of the amino acids has been effected. The smaller variety of amino acids compared with the structures of peptides makes HPLC modes are available to the researcher cation exchange chromatography (CEC), and reverse phase chromatography (RPC). Reverse phase chromatography is most suitable for the separation of amino acid derivatives as for amino acids.
The choice of HPLC is limited by the choice of the derivatization agent and / or the decision to use pre-and post-column derivatization, in fact, some of the most interesting discoveries made in the HPLC separation of amino acid resides in the development and optimization of new derivatization agents. Very fast reactions and derivatization with fluorescamine and ortoftaldehido (OPA) are being used increasingly to classical ninhydrin method.4. Derivatization reagents.
The problem of the derivatization of amino acids is concentrated in the selective and sensitive detection thereof. The amounts of available sample are very small, whether proteins whose structure is to be determined or research samples in the food science field, so that the derivatization reaction should allow the detection in the range pmol.
The derivatization may be performed prior to separation. Should occur quantitatively to produce simple products for each amino acid, the reagent should not interfere. Derivatization is likely to generate products that are more similar to each other, in any case, high selectivity of separation of chromatographic systems which rarely involves no problems. Precolumn derivatization can be carried out automatically immediately before the separation.
Derivatization after removal generally requires a higher instrumental complexity. Commercial instruments often need to be optimized to achieve higher sensitivity of detection.
Ninhydrin (triketohydrindene hydrate) reacts with amino acids in acidic medium (pH between 3 and 4) and producing hot ammonia, carbon dioxide and a bluish purple complex. All primary amino acids are the same complex after reaction with ninhydrin, making this agent unsuitable for precolumn derivatization. Most columns of cation exchange HPLC used for amino acid analysis with postcolumn ninhydrin based resins sulfonate still employing polystyrene / divinylbenzene.
The first automatic systems for the analysis of amino acids described in 1958 based on the amino acid separation by cation exchange chromatography and subsequent ninhydrin reaction remains today as the most reliable method for the analysis of amino acids in peptides and proteins. Commercial analyzers based on this methodology are available from companies such as Beckman and Pharmacia. At high temperatures (>= 100 C), all the primary amino acid react with two molecules to form a chromophore ninhydrin (Ruthermann purple) with maximum absorption at 570 nm.
In the quantification of individual amino acids may be used the molar absorption coefficient of the colored compound, although its value varies from one amino acid to another and should be determined for the particular conditions of the analysis. However, one must choose a reference value to be used for the quantification of total amino acids in a mixture.
The ninhydrin color reaction of the used analytical as well as in the display of the amino bands after separation by electrophoresis or chromatography. The reagent used in these circumstances is generally prepared dissolved in ethanol when added 2.4-6colidina, color differences between each spot help identify individual amino acids.
The OPA was originally introduced as an alternative reagent to the post-column ninhydrin derivatization and provided a significant increase in sensitivity. Amino acids react with the OPA in the presence of a strong reducing, under alkaline conditions (pH 9-11), giving a fluorescent compound. The reverse phase separation followed by fluorescent detection method of detection is a rapid, sensitive and selective for all amino acids with primary amine groups. Peptides are less reactive than -amino acids and secondary amines do not react.
While the OPA was originally applied only for derivatization postcolumnas is now widely used as an agent for precolumns. While the separation of amino acids following a precolumn derivatization is carried out by reverse phase chromatography, both reverse phase chromatography and cation exchange chromatography may be used when the OPA is the agent in the post-column derivatization.
A drawback of OPA resides in the relative instability of its derivatives, may added to the quantization problems, although this is no great difficulty in the art of post-column derivatization. However, precolumn and from the lowest point of pH 7.2 to pH 9.5 typical reaction, the products must be immediately applied to a chromatographic system. Lowering these two buffers the reaction pH and slightly serves to stabilize the reaction products. The major disadvantage of OPA is does not react with secondary amines (proline and hydroxyproline) although this can be solved by oxidation with chloramine-T or hypochlorite. A strategic combination of using OPA FMOC (9-fluorenylmethyl chloroformate) has recently been introduced to address this deficiency.
The OPA method is used specifically with precolumn derivatization with amino acid analysis is more sensitive because it takes less time than other methods HPLC.
The fluorescamine was also introduced as a possible alternative to postcolumnas ninhydrin derivatization. All primary amines react with fluorescamine in basic medium (pH 9-11) give a fluorescent product with increased detection sensitivity increased to ninhydrin, although fluorescamine itself has no fluorescence. The fluorescent product is unstable in aqueous solution and the reagent must be prepared in acetone. Secondary amines do not react, unless previously converted into primary amines, which can be done with N-chlorosuccinimide, or chloramine-T hypochlorite. Although the reagent is of interest for its rapid rate of reaction with amino acids at room temperature, the reaction is not as sensitive as ninhydrin.
Phenyl isothiocyanate (PITC).
PITC, also known as Edman reagent has been widely used for sequencing polypeptides and proteins and was introduced to amino acid analysis at the beginning of the eighties. It is currently the most widely used agent for precolumns derivatization followed by reverse phase chromatography, analysis of amino acids, amino acid mixtures used for a wide variety of sources.
Instead of being analyzed as derivatives of PTH (feniltiohidantoin), as occurs during peptide or protein sequencing, amino acid derivatives are analyzed as PTC (feniltiocarbamol). Thus, at alkaline pH, primary and secondary amino acids produced PTC derivatives, which can be registered by UV absorbance (240-255 nm) with a detection limit in the range of 5 to 50 pmol. All amino acids are mono-PTC, except lysine and cysteine, which produce double PTC forms. The products are relatively stable, although some conversion to the PTH derivative may occur if the pH is not properly controlled.
Although PITC methodology can be considered superior to other techniques in many aspects, has a major disadvantage that some PTC derivatives of amino acids are strongly affected by the presence of some salts, divalent cations, metals and ions tampon. Thus, while the ammonium acetate, sodium chloride and sodium borate have been found to have no effect in PTC amino acids, other salts, such as sodium phosphate and sodium bicarbonate if they have it. Besides contamination by trace amounts of metal ions has been found to reduce the formation of PTC amino acids. Adding EDTA even better yields were achieved for derivatization.
Dansyl chloride and dabsyl chloride.
Dansyl chloride (chloride, 1-dimethylaminonaphthalene-5-sulfonyl) was initially introduced to the N-terminal analysis of the amino acids in peptides and proteins, for a purpose which is still employed. Reacts with primary and secondary amines to produce fluorescent derivatives. Its application suffers from the presence of numerous side products and the formation of multiple derivatives of certain amino acids. Added to this is the requirement of a long reaction time. Thus, although the dansyl chloride is frequently used by the researchers to precolumn derivatization followed by reverse phase chromatography, this method has never been widely accepted for automated amino acid analysis.
Dabsyl chloride (dimetilaminoazobencenosulfonilo chloride) is a closely related agent dansyl chloride. It was introduced and developed by Chang and colleagues, for the detection of amino acids in the visible range and pmol level. Reacts with primary and secondary amino acids to produce derivatives having a large absorbance in the visible range. The reaction is performed at about 70 C and a pH of 8 to 8.5, yielding highly stable derivatives 10 or 12 minutes. Form mono and bis derivatives of lysine, tyrosine and histidine.
Despite the advantages of dansyl chloride as derivatizing agent, the technique of dabsyl chloride has a limited tolerance to the presence of salts, and it is not yet clear how the detection would suffer high levels of sensitivity (range 10 to 20 pmol) in the presence of metals, salts and other contaminants.
Although not used widely in amino acid automatic analysis ninhydrin, OPA and the PITC, a commercial adaptation of dabsyl chloride method is available from Beckman Instruments.
9-Fluoroenilmetil chloride chloroformate (FMOC-Cl).
The FMOC-Cl reacts with amino groups to form highly fluorescent derivative carbamados and stable. This highly reactive agent, originally and still used as amino protective group in peptide synthesis, was first applied in a precolumn derivatizing agent for amino acid analysis. The FMOC-Cl reacts with all amino acids at alkaline pH and ambient temperature to produce amino acid derivatives that have a highly stable fluorescence comparable to OPA derivatives. They can form mono-and bis-derivatives with histidine, tyrosine and lysine. The relative proportions of these derivatives vary depending on the pH and the proportion of reagent and amino acid.
During the reaction with FMOC-Cl, hydrolysis products formed have reactive fluorescence similar to amino acid derivatives. These products interfere with the separation by reverse phase chromatography unless removed by extracting the organic solvent before applying the sample to the column of reverse phase chromatography. An automated version of this derivatization procedure has been developed and commercialized.
Foldi Betnr and solved the problem of interference by the large peak eluting FMOC-Cl and their hydrolysis products, FMOC-OH, which are eluted in the same region chromatographic many of FMOC amino acids by derivatization excess volume FMOC-Cl with a hydrophobic amine. The resulting derivative is eluted later in the chromatogram after all FMOC amino acids.
A combination of OPA / FMOC has been described, which uses a commercially available analyzer (Hewlett-Packard Amino Quant). The primary amino acid reaction is carried out initially by OPA followed by reaction with FMOC-proline and hydroxyproline. The determination of the fluorescence for the primary amino acid derivatives OPA and FMOC-proline is then conducted to appropriate wavelengths.
7-chloro, 7-fluoro-4-nitrobenz-2-oxa-1 ,3-diazole (NBD-Cl and NBD-F).
Derivatisation reagents based nitrobenzofurazan structure, NBD NBD-Cl and-F, have proven useful for specific applications in the analysis of all amino acids.
The NBD-Cl was introduced by Ghosh and Whitehouse fluorignico as an agent for amines, including amino acids and subsequently Rot as a useful tool for the analysis of amino acids, in particular their enhanced reactivity with secondary amines. Since then, this reagent has occasionally been employed for the detection of amino acids, with particular emphasis on the detection of proline and hydroxyproline. NBD-Cl has been applied to the derivatization in pre-and post-column, with a detection limit of 1 nmol latter for proline and hydroxyproline and a detection limit in the first somewhat less. Although NBD-Cl is stable and allows sensitive detection of amino acids, reacts with amines more slowly and has not had widespread use as general analytical agent for amino acids.
NBD-F is more reactive than its chloro analogue, allowing derivatization faster, accompanied by a large fluorescence for proline and hydroxyproline. Similar to NBD-Cl, NBD-F has been applied both to as postcolumn derivatization precolumn. Detection limits are defined for the pre-column in the range of 5-15 fmol for most amino acids. The NBD-F has not been used widely as an analytical method for amino acids, although some researchers routinely employ this reagent.
Other derivatives of the isothiocyanate reagents.
The 4′-N, N-dimethylamino-4-azobencenoisotiocianato (DABITC) has been successfully used in manual sequencing and terminal nitrogen analysis of peptides and proteins. Type Edman degradation of peptides and proteins made by this colored tiohidantoin agent produces derivatives (DABTH) which can be separated by reverse phase chromatography. The degradation method generally comprises a double coupling with DABITC first and then with PITC. The 4-N, N-homologous dimetilnaftilazobenzeno DABITC (DANABITC) has also been successfully applied to a lesser extent than DABITC.
Difenilindenonilisotiocianato (DIITC) has been studied to be used when mass spectrometry is coupled by chemical ionization with previous identifications by reverse phase chromatography. Detection tiohidantoin derivatives (DITH) is carried out by UV determination (in the range below pmol). The electrochemical detection is an alternative utensil.
Other reagents precolumn derivatization isothiocyanates are 4 – (t-Butyloxycarbonylaminomethyl) phenyl isothiocyanate (BAMPITC, fluorescent detection) trimetilsisil for sequence analysis isothiocyanate terminal carbon (UV detection) and pN, N-dimethylaminophenyl isothiocyanate (DMAPI) as an electrochemical instrument to amino acid analysis.
Other reagents are continually being developed and used for amino acid detection. Some have very specific and limited applications, while others are treated as potential competitors for broad applications in automated systems. Such reagents include 2,4,6-trinitrobenzenesulfonic acid (TNBS) and its analogue 2,4-dinitrobenzene (DNBS), N, N-diethyl-2 ,4-dinitro-5-fluoroaniline, fluoro-2 ,4-dinitrobenzene , 1,1-difenilbrico acid and 1-fluoro-2 ,4-dinitrophenyl-5-L-alanine amide (Marfey’s reagent). The fluorescent detection is possible when using reagents such as pentane-2 ,4-dione / formaldehyde 9-antrildiazometano, laril chloride, 2,3-naphthalene-dicarboxialdehido, -naftilcarbamato succinimide, 3-benzoyl-2quinolina-carboxaldehyde, 9 hidroximetilantraceno and fluorescent isothiocyanate-and 1-naphthyl isocyanate. A chemiluminescent reagent, 4-isocianatoftalhidrazida, has also been investigated. With the exception of pentane-2 ,4-dione / formaldehyde, where the reagent was used in postcolumn derivatization followed by ion exchange HPLC of amino acids, all others were used for derivatization prior to reverse phase chromatography.
In the following table to compare the different characteristics of the major derivatization reagents for the determination of amino acids:
Sample preparation. The reaction to form the same chromophore derivatizing with primary amines to minor therefore samples must be free from salts and lipids take place before the hydrolysis and derivatization steps. The major cause of low yields in the derivatization reactions is the presence of contaminants in the sample, such as non-volatile salts, detergents and lipids. Salts and buffers both interfere in the subsequent hydrolysis and derivatization and chromatography distort extra added chromatogram peaks.
The salts can be removed from the proteins and peptides using volatile buffers and reverse phase HPLC, precipitation, dialysis or gel filtration columns.
5. Pre-column derivatization versus post-column
The researchers opinions about the relative merits of derivatization before or after HPLC, amino acids will be determined by the specific application requirements. Factors such as the sensitivity required to detect, sample available, the type of sample, sample source, scan speed and reproducibility even economic considerations influence the choice of the derivatization pre-or post-column amino acid.
Post-column. Derivatization following cation exchange chromatography of amino acids has several advantages, as long as the speed and instrumentation produce reproducible reaction temperatures. Furthermore, for the derivatization is not necessary to completely and stability problems arising is insignificant. Other advantages are that the automation is easy and the time consumed and loss due to the sample preparation are unnecessary. Detectors can also be used in series. A lot of experience has been gained from traditional cation exchange method and staining with ninhydrin, which has proven to be reliable, accurate and capable of excellent separation of complex mixtures of amino acids and their metabolites.
A disadvantage with a conventional HPLC instruments is that they are more complex than necessary for the pre-column method. The post-column derivatization requires the addition of one or more pumps for derivatizing reagents and solvents. It also requires the addition of a post-column reactor, which lead to additional band broadening, and therefore, a decrease in detection sensitivity. The column elution is continuously diluted by the derivatizing reagent, causing a decrease in detection sensitivity. Thus, the maximum achievable sensitivity by post-column derivatization is lower than in the case of the precolumn.
Pre-column derivatization. This, coupled with reverse phase chromatography instead of cation exchange chromatography, was originally introduced as a response to increasing demand for higher speed and higher sensitivity analysis. The advantages of the methodology of the pre-column derivatization include simplicity, speed and high sensitivity. Therefore, if high sensitivity is required for detection, a fluorescent reagent combined with pre-column derivatization method is most suitable.
A disadvantage is the need to ensure complete reaction of the derivatizing reagent and the possibility of interference with the separation of excess reagent, the reaction medium or the production of various derivatives of one component. Moreover, the stability of the derivative may be an important factor during the pre-column derivatization, the delay between injection and derivatization becomes critical to the results.
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