High performance liquid chromatography coupled with chemometric methods
Liu Guangjun (Department of Chemistry, Jining Teachers College, 272025, Jining City, Shandong Province)
Abstract: The DL-amino acid enantiomers were resolved by conventional reversed-phase high performance liquid chromatography using phthalaldehyde and N-acetyl-L cysteine ​​as pre-column chiral derivatization reagents for baseline separation. The chromatographic peak is calculated and analyzed by chemometric method, so as to achieve the purpose of simultaneous quantitative determination of multiple amino acid enantiomers.
Key words: amino acid; 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 been a hot research topic at home and abroad. In the field of chiral separation, high efficiency Liquid chromatography (HPLC) has been the most widely used method. There are two main methods for separating chiral compounds by high performance liquid chromatography : one is direct separation, which is the direct separation of chiral compounds by chiral stationary phase.æ˜ ä½“. Wang Yali [1] and other used cellulose-tris(3,5-dimethylphenylcarbamate)
(CDMPC) The chiral column splits three racemic amino acid derivatives in normal phase mode. The other is indirect separation, which is currently mainly pre-column derivatization--pre-column derivatization of chiral compounds. The enantiomers were converted to diastereomers, and the separation analysis was performed using a conventional column. Lu Haitao [2] discussed the effect of mobile phase on the separation of DL-amino acids by pre-column derivatization.
This paper mainly discusses the separation of enantiomers of various amino acids by pre-column derivatization and the introduction of chemometric methods into the overlapping peaks of serine enantiomers. The enantiomers of various amino acids can be quantitatively analyzed at one time. O-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 derivatizing agent together with OPA.
1 Experimental part
1. 1 reagents and instruments
DL-serine (Shanghai Lizhu Dongfeng Biotechnology Co., Ltd.); L-serine, beta alanine, DL-alanine, L-alanine, DL-phenylalanine, L-phenylalanine, DL -proline, L-valine, boric acid, potassium chloride, sodium hydroxide, sodium acetate (China Pharmaceutical Group Shanghai Chemical Reagent Company); o-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 of analytical grade.
American Aglient HP1100 High Performance Liquid Chromatograph (DAD Detector), ChemStation ChemStation. Agilent 8453 UV
- Vis spectrometer.
1. 2 sample pretreatment [5]
Each amino acid sample was formulated into an aqueous solution having a concentration of about 0.01 M. Preparation of boric acid buffer: boric acid (0.0 M), sodium hydroxide (0.0 M) and water were prepared in a volume ratio of 50:45:5. Boric acid buffer with a pH of 9.3. Formulation of the derivatizing agent: 53.3 mg of OPA was dissolved in 50 mL of methanol to obtain OPA methanol solution. NAC was dissolved in boric acid buffer (0. 00286 M). 12 mL of OPA methanol was taken. Solution, 10mL NAC boric acid solution, add 3 mL boric acid buffer to 25 mL to obtain OPAPNAC derivatizing agent.
1. 3 Derivatization reaction 0.1 ml of amino acid and 5 mL of OPAPNAC derivatizing agent were thoroughly mixed for 5 min and then filtered for injection analysis.
1. 4 chromatographic conditions
ZORBAX Eclipse XDB - C8 column (4.6 mm 3 150mm, 5μm). Different ratios of methanol and 0.05 M sodium acetate aqueous solution are mobile phase, flow rate 1 mL·min -1 , injection volume 20 μL. All chromatographic separations Performed at room temperature, the on-line detection wavelength is 334
The nm and DAD detection wavelength range is 190-400 nm. Non-negative matrix factorization (NMF) calculates the intercepted data wavelength range from 320 nm to 390 nm.
1. 5 Data processing method This experiment uses non-negative matrix factorization (NMF) [6] to calculate the pure spectrum of two mixed components. Non-negative matrix factorization is in "non-negative"
A new matrix decomposition method under constrained constraints, the basic idea is to decompose the non-negative matrix V into two non-negative factor matrices W
The multiplication update formula is used in the H.NMF algorithm (see equations (1) and (2)), so the decomposition result can be guaranteed to be “non-negative†without using other constraints.
2 Results and discussion
2. The separation of the amino acid enantiomers from the derivatizing agent to form the isoindole product [7], the reaction equation is shown in Figure 1. It can be seen from the spectrum obtained by UV measurement.
Such derivatives have a maximum absorption at 230 nm and 334 nm (see Figure 2). However, since the 230 nm is more susceptible to interference, in addition to recording full-wavelength data, 334 nm is selected as the detection wavelength in the chromatographic experiment. The amino acid peaks mentioned below are the chromatographic peaks of the derivatives obtained after the above derivatization.
Fig.1 Derivative reaction equation of DL-alanineFig.2 UV absorption experiment of 2β alanine derivative at 190 nm～400 nm shows that DL is removed under the leaching condition of methanol: sodium acetate solution at 30:70 - The enantiomers of DL-alanine, DL-valine and DL-phenylalanine can be separated by baseline, but the two pairs of DL-valine are separated by partial overlap of the two enantiomers of serine. The peak time of the body is 17 min and 25 min. 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. Adjust the ratio of mobile phase to 45:55. Although the enantiomers of DL-valine and DL-phenylalanine can elute peaks within 10 min, DL-serine and DL-alanine will be made. The acid is completely or partially overlapping.
Considering the excessive retention of DL-valine and DL-phenylalanine, 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 rapid peak. The elution scheme is as follows: keep methanol: sodium acetate solution at 30:70 for 0-6 min, and linearly change to 45:55 at 6-7 min. Maintain 45:55 after 7 min
No change. The characterization of each pair of enantiomers was determined by internal standard method using a left-handed optically pure standard. β-alanine has no chirality and no enantiomers.
There is only one chromatographic peak. Figure 3 is a chromatogram derived from a mixture of DL-serine, DL-alanine, beta alanine, DL-valine and DL-phenylalanine.
Figure 3 Chromatogram of four racemic amino acids and beta alanine
2. 2 Use of Spectral Analytical Method As can be seen from Figure 3, despite the gradient elution, there is still a pair of overlapping peaks in the spectrum - the two enantiomers of serine. In this case,
If the system is to be quantitatively analyzed, it is necessary to know the actual peak area of ​​the two components. We use a non-negative matrix factor analysis to resolve the pure spectrum of the two components (see Figure 4), but this result also exists with the actual pure spectrum. The coefficient relationship, ie AXD-Ser + BXL-Ser = YDL-Ser . To obtain the actual pure spectrum of the two components, we use the 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 the respective components to determine the actual peak area of ​​the two components.
2. 3 Quantitative analysis of each enantiomer
2. 3. 1 The establishment of the standard curve establishes a standard curve with the peak area or peak height of the single enantiomer standard at different concentration values.
In the experiment, L-alanine and L-phenylalanine were used as two standards, and L-alanine corresponds to the chromatographic condition of methanol: sodium acetate = 30:70, L-
Phenylalanine corresponds to the chromatographic conditions of methanol: sodium acetate = 45:55. Linearly fit the peak area or peak height to obtain the standard curve equation (see Figures 6 and 7). This equation can be used to determine the mixed sample. The concentration of the two standard components.
2. 3. 2 Relative concentration prediction of other components Under the same chromatographic conditions, the content ratio of each component in the system is linear with the ratio of the peak area of ​​the chromatographic peak. See the ratio of the peak area of ​​each component and the area ratio of the occupied area. Table 1, wherein the single peak area of ​​D-serine and L-serine is calculated according to the above stoichiometry method.
3 Conclusions In this paper, the pre-column derivatization method was used to achieve the resolution of four kinds of amino acid enantiomers by conventional reversed-phase high performance liquid chromatography, which achieved better separation effect and realized multi-component quantitative analysis. The washing scheme is also relatively simple. The use of computational methods to resolve overlapping peaks reduces the need for chromatographic peak resolution, which is useful for analyzing more complex sample systems.
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