Liu Guangjun (Department of Chemistry, Jining Teachers College, 272025, Jining City, Shandong Province)
Abstract: Using phthalaldehyde and N-acetyl-L cysteine ​​as pre-column chiral derivatization reagents, the DL-amino acid enantiomers were resolved using conventional reversed-phase high-performance liquid chromatography. Chromatographic peaks are calculated and analyzed using chemometric methods to achieve the purpose of quantitative determination of multiple amino acid enantiomers simultaneously.
Keywords: amino acids; chiral separation; high performance liquid chromatography; derivatization
In recent years, amino acid analysis has been widely and importantly applied in biochemistry, pharmacy and clinical research. The separation of amino acids, especially the separation of amino acid enantiomers, has always been a research hotspot at home and abroad. In the field of chiral separation, high efficiency Liquid chromatography (HPLC) has always been the most widely used method. At present, there are two main methods for separating chiral compounds by high-performance liquid chromatography: one is the direct separation method, which is the direct separation of chiral compound pairs using a chiral stationary phase. Enantiomer. Wang Yali [1] et al. Used cellulose-tris (3,5-dimethylphenylcarbamate)
(CDMPC) The chiral column resolves three racemic amino acid derivatives in normal phase mode. The other is the indirect separation method, which is currently the pre-column derivatization method-the pre-column derivatization of chiral compounds Chemical conversion, the enantiomers are converted into diastereomers, and then the separation analysis is completed using a conventional column. Lu Haitao [2] has discussed the effect of mobile phase when DL-amino acids are resolved by pre-column derivatization.
This article mainly discusses the use of pre-column derivatization to separate enantiomers of multiple amino acids, and introduces stoichiometric methods into the analysis of overlapping peaks of serine enantiomers. Phthalaldehyde (OPA) and N-acetyl-L-cysteine ​​(NAC) [2,3]. NAC is a chiral thiol, other chiral thiols such as N-acetyl-D-penicillium Amine (NAP), N-isobutyryl-L-cysteine ​​(IBLC), N-isobutyryl-D-cysteine ​​(IBDC) [4] can also be used as a derivative with OPA.
1 Experimental part
1. 1 Reagents and instruments
DL-serine (Shanghai Lizhu Dongfeng Biotechnology Co., Ltd.); L-serine, β-alanine, DL-alanine, L-alanine, DL-phenylalanine, L-phenylalanine, DL -Valine, L-valine, boric acid, potassium chloride, sodium hydroxide, sodium acetate (China Pharmaceutical Group Shanghai Chemical Reagent Company); phthalaldehyde (hereinafter referred to as OPA, China Pharmaceutical Group Shanghai Chemical Reagent Company) ; N-acetyl-L-cysteine ​​(hereinafter referred to as
NAC, Lancaster); Methanol (HPLC grade, Merck); High-purity water. All reagents except methanol and water are analytically pure.
American Aglient HP1100 high performance liquid chromatograph (DAD detector), ChemStation ChemStation. American Aglient8453 UV
-Vis spectrometer.
1. 2 Sample pretreatment [5]
Each amino acid sample is prepared into an aqueous solution with a concentration of about 0.01 M. The preparation of boric acid buffer solution: boric acid (0.01 M), sodium hydroxide (0.01 M) and water are prepared according to a volume ratio of 50: 45: 5 Boric acid buffer with pH 9.3. Preparation of derivatizing agent: dissolve 53.3 mg OPA in 50 mL of methanol to obtain OPA methanol solution. NAC is dissolved in boric acid buffer (0.00286 M). Take 12 mL of OPA methanol Solution, 10mL NAC boric acid solution, add 3mL boric acid buffer to 25mL to obtain OPAPNAC derivatizing agent.
1.3 Derivatization reaction Mix 0.1 mL of amino acid with 5 mL OPAPNAC derivatization agent thoroughly for 5 min, then filter and analyze.
1. 4 chromatographic conditions
ZORBAX Eclipse XDB-C8 column (4.6 mm 3 150mm, 5μm). Different proportions of methanol and 0.05 M sodium acetate aqueous solution as mobile phase, flow rate 1 mL · min-1, injection volume 20μL. All chromatographic separations are Performed at room temperature, the online detection wavelength is 334
nm and DAD detection wavelength ranges from 190 to 400 nm. Non-negative matrix factorization (NMF) calculates the intercepted data wavelength range from 320 nm to 390 nm.
1. 5 Data processing methods This experiment uses non-negative matrix factorization (NMF) [6] to calculate the pure spectrum of the two mixed components. Non-negative matrix factorization is in "non-negative"
A new method of matrix decomposition under restricted constraints, its basic idea is to decompose the non-negative matrix V into two non-negative factor matrices W
The H.NMF algorithm uses the multiplication update formula (see formulas (1) and (2)), so it is possible to ensure that the decomposition result is "non-negative" without using other restrictions.
2 Results and discussion
2.1 Separation of amino acid enantiomers The reaction of amino acids with derivatizing agents produces isoindole products [7], the reaction equation is shown in Figure 1. The spectrum obtained by UV measurement can be seen,
Such derivative products have an absorption maximum at 230 nm and 334 nm (see Figure 2). However, because 230 nm is more susceptible to interference, in addition to recording the full wavelength data in the chromatography experiment, 334 nm is selected as the detection wavelength The amino acid chromatographic peaks mentioned below are the chromatographic peaks of the derivatives obtained after the above derivatization.
Fig.1 Derivative reaction equation of DL-alanineFig.2 The ultraviolet absorption experiment results of 2βalanine derivatives at 190 nm ～ 400 nm show that under the leaching condition of methanol: sodium acetate solution of 30:70, except DL -The two enantiomers of serine partially overlap, the enantiomers of DL-alanine, DL-valine, and DL-phenylalanine can be baseline separated, but the two enantiomers of DL-valine The peak time of the body is 17 min and 25 min, and the peak time of the two enantiomers of DL-phenylalanine is 38 min and 43 min. Under this separation condition, not only the mobile phase is wasted, but also the peak shape is not ideal. If Adjusting the ratio of mobile phase to 45:55, although the enantiomers of DL-valine and DL-phenylalanine can be eluted within 10 minutes, it will make DL-serine and DL-alanine The acids overlap completely or partially.
Considering that the retention of DL-valine and DL-phenylalanine is too strong, a simple gradient elution was used in the operation.After all the first three amino acids peaked, the mobile phase ratio was changed to make DL-valine And DL-phenylalanine peak quickly. The elution scheme is as follows: keep the methanol: sodium acetate solution unchanged at 30:70 within 0 to 6 minutes, and the ratio changes linearly to 45:55 from 6 to 7 minutes. 45:55 after 7 min
The same. The characterization of each pair of enantiomers is determined by the internal standard method using pure optical standard of left-handed rotation. Î’-alanine has no chirality and no enantiomers.
There is only one chromatographic peak. Figure 3 shows the chromatogram after DL-serine, DL-alanine, β-alanine, DL-valine, and DL-phenylalanine mixture.
Figure 3 Chromatogram of four racemic amino acids and β-alanine
2. The use of spectral analysis method can be seen from Figure 3, despite the gradient elution, there are still a pair of overlapping peaks-two enantiomers of serine in this spectrum. In this case,
If you want to quantitatively analyze this system, it is necessary to know the actual peak area of ​​the two components. We use non-negative matrix factor analysis to resolve the pure spectrum of the two components (see Figure 4), but there is still a difference between this result and the actual pure spectrum. The coefficient relationship, that is, AXD-Ser + BXL-Ser = YDL-Ser. To obtain the actual pure spectrum of the two components, we use least squares regression (LSR) to calculate the coefficients A and B, and the results are as follows ( (See Figure 5): A = 72.59; B = 75.98. This coefficient is multiplied by the pure spectrum of each component to be the actual peak area of ​​the two components.
2. 3 Quantitative analysis of each enantiomer
2.3.1 The establishment of a standard curve The chromatographic peak area or peak height of a single enantiomer standard at different concentration values ​​is used to establish a standard curve.
In the experiment, L-alanine and L-phenylalanine were used as two standards. L-alanine corresponds to the chromatographic conditions of methanol: sodium acetate = 30:70, L-
Phenylalanine corresponds to the chromatographic conditions of methanol: sodium acetate = 45:55. The linear fitting of the peak area or height with the concentration yields a standard curve equation (see Figures 6 and 7). This equation can be used to determine mixed samples The concentration of the two standard components.
2.3.2 Prediction of relative concentration of other components Under the same chromatographic conditions, the content ratio of each component in the system is linearly related to the ratio of its chromatographic peak area. The peak area ratio of each component and the area area ratio results are shown in Table 1, where the single-peak area of ​​D-serine and L-serine is calculated according to the above stoichiometric method.
3 Conclusion In this paper, the pre-column derivatization method was used to achieve the resolution of the four amino acid enantiomers using conventional reversed-phase high performance liquid chromatography, which achieved a good separation effect and realized the quantitative analysis of multi-components. Washing scheme is also relatively simple. Using calculation methods to resolve overlapping peaks relatively reduces the requirements for chromatographic peak resolution, which is very helpful for analyzing more complex sample systems
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