A Full Introduction to Amino Acid Analysis Methods (Part

Three)

1. Dansyl-C1 Derivative Method

The Dansyl-C1 derivatization method is relatively simple. You only need to add
Dansyl-Cl to a potassium bicarbonate buffer solution (pH = 9.5) in which amino acids
are dissolved in advance, and then complete the reaction at room temperature for 35
to 50 minutes.

The yield of this reaction derivative depends on the ratio of Dansyl-C1 to amino acid.
Tapuhi et al. suggested that the most appropriate ratio was 5: 1 to 10: 1 (mol /
mo1) by examining and optimizing the derivatization conditions.

The advantages of Dansyl-C1 as an amino acid derivatizing agent are: Dan-sy-Cl and
all amino acids, including imino, can react, and the derivatives are relatively stable,
which can generally be left for 12 to 24 hours. The disadvantages of this method
are: poor reactivity and slow reaction speed; Dansyl-C1 hydrolysate Dansyl-SO H can
also emit strong fluorescence and interfere with the determination; the derivative
product is more sensitive to ultraviolet light, and the reaction should be performed
under dark conditions ; Easy to generate multi-level derivative products, such as
reaction with histidine, lysine and cystine can generate secondary derivatives, which
will bring some trouble to separation and determination. In addition, if the reaction
temperature is higher than 45 ° C, the rate of derivative formation will increase, but
the amount of dansylamide and other by-products will also increase rapidly, which
will reduce the yield of the derivative.

2. Dinitrofluorobenzene (FDNB) derivatization method

The conditions for the derivatization reaction of FDNB with amino acids are easy to
control, and its derivative product 2,4-nitronitroamino acid has good stability. The
response value does not change significantly after being stored in the dark at 4 ° C
for one month. Brings great convenience to quantitative determination.

Due to the introduction of the nitrobenzene ring, the derivative produced by this
reaction can generate strong ultraviolet absorption, and the detection sensitivity can
reach Drool level. In addition, the derivative has a short chromatographic separation
time, and the accuracy and repeatability of the analysis result are relatively good.
Due to the strong binding of FDNB to the amino group, this derivatization product is
quite stable and is an ideal reagent for pre-column derivatization of amino acids.

The main disadvantage of this method is that the retention time of the byproduct of
the derivatization reaction, dinitrophenol, and the main derivative are similar, which
will interfere with the separation process. The solution is to control the amount of
derivatization reagent FDNB and improve the separation conditions.

3. 1-Fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDNPAA) Derivative

Method

FDNPAA is a new type of chiral derivatization reagent. It is used for derivatization

and separation of DL-amino acids. The derivative product of this reaction is stable
within 5 hours. 20 amino acids including proline and photoline can be well separated
in 10 min, and the limit amount that can be detected by the 340nm UV detector
reaches 50 X 10 tool. This method does not undergo racemization during the
derivatization process, and the reaction rate is consistent, the separation effect is
good, and it can achieve baseline separation with other materials, so the recovery
rate is high, and the stability, precision, reproducibility and versatility are good; The
main disadvantages are that the derivatization reagent is more expensive and the
derivatization time is longer.

4. Sulfonyl chloride dimethylamine azobenzene (DABS-CI) derivatization

method

The characteristic of this method is that the detection sensitivity of the DABS-C1
amino acid derivative at 430 nm in the visible light region can reach 1 × 10 mol,
which is similar to the sensitivity of the fluorescence detection method of 0.5 × 10 2
mol. Absorb the interference of impurities. In addition, the derivatization method is
simple and fast, and the derivatives are extremely stable, which can be maintained
for more than one month at room temperature in sodium carbonate solution.

Earlier DABS-C1 derivatization methods could not be used for quantitative analysis of
unknown samples due to too many derivatives. Later, Chang et al. (B) controlled the
ratio of DABS-C1 to the amino acid molecule to 4: 1 to 8: 1 (mol / mo1), and
improved the derivatization conditions so that only one derivative of the amino acid
was generated, which better solved this problem. Limits can reach sub-pmol levels.

5. 6-aminoquinolinyl-N-hydroxysuccinimidyl carbamate (AQC)

derivatization method

AQC reacts with both primary and secondary amino acids, derivatizes quickly, the
derivatives are stable, and the UV detection sensitivity is high (the detection limit is
lower than the sub-pmol level).

In addition, the method not only has the precision and accuracy comparable to ion
exchange chromatography, but also is not affected by the sample matrix, a large
number of electrolytes, vitamins and trace elements. It is especially suitable for the
analysis of amino acids in natural biological samples, food and feed. The main
problem with this method is that the 6-aminoquinoline produced after AQC hydrolysis
has strong UV absorption, and a large reagent peak is formed before the peak of the
conventionally hydrolyzed amino acid derivative with tailing phenomenon, which
interferes with Determination of amino acids such as aspartic acid and serine.
However, this problem can be solved by optimizing the mobile phase pH and
gradient elution procedure.

6. 2,4-Dinitrochlorobenzene (CDNB) Derivative Method

CDNB has a similar nitrophenyl group as FDNB, so its pre-column derivatization and
chromatographic analysis conditions are similar to FDNB method.

Reaction of CDNB with amino acids Compared with FDNB method, CDNB method has
the characteristics of cheaper and easier derivatization reagents, stable physical and
chemical properties, and convenient and simple preparation of samples. It has been
reported that a gradient elution method using a C 18 column and an acetonitrile-
acetate buffer system can effectively separate 17 mixed amino acids and derivatizing
agent hydrolysates.

Amino acids derived by this method are generally separated on a bonded C 18 column
according to the liquid-liquid distribution principle. Chromatographic mobile phases
are mostly acetate or phosphate buffer, and acetonitrile, methanol, or
tetrahydrofuran are used as regulators. Because the amino acid derivative still
retains the characteristics of the amphoteric compound, in addition to changing the
regulator, it can also achieve the ideal separation by adjusting the pH, ionic strength,
and column temperature of the buffer. Of course, the column type, mobile phase,
and amino acid elution time and order chosen for different derivatives vary. This
method can be used to analyze amino acids in samples such as protein hydrolysates,
physiological fluids, and food.

Conclusion

This article comprehensively discusses some analytical methods of amino acids

(chemical analysis, spectral analysis, electrochemical analysis, capillary
electrophoresis, and chromatography), and focuses on the pre-column derivatization
of amino acids-reversed-phase high-performance liquid chromatography (The
research progress of RP-HPLC) is elaborated in detail, and the experimental effects
of different derivatization reagents are discussed and compared, which provides a
method reference for daily amino acid analysis.