sample pretreatment

Instrument configuration and chromatographic conditions

Spectrum and data

3.1 Separation spectrum and data of mixed standard solution of alpha lactalbumin and beta lactalbumin

Figure 1 Separation spectrum and data of a mixed standard solution of alpha lactalbumin and beta lactalbumin (100 μ g/mL)(Click to view large image)

As shown in Figure 1, the chromatographic peak shapes of α - lactalbumin and β - lactoglobulin are good, the column efficiency is excellent, and the separation degree is better than the standard. The retention time of alpha lactalbumin is 21.900 min, the asymmetry factor is 0.98, and the theoretical number of trays is 321137; The retention time of β - lactoglobulin B is 24.747 min, the asymmetry factor is 1.01, and the theoretical number of trays is 352451; The retention time of β - lactoglobulin A is 25.467 min, the asymmetry factor is 1.00, and the theoretical number of trays is 373359. The separation degree between alpha lactalbumin and beta lactalbumin B is 17.77, and the separation degree between beta lactalbumin B and beta lactalbumin A is 4.33.

3.2 Single standard solution localization and superposition spectra of α - lactalbumin and β - lactoglobulin

Figure 2: Overlay spectra of single standard solutions of alpha lactalbumin and beta lactalbumin (click to view larger image)

The single label localization chromatograms and spectra of alpha lactalbumin and beta lactalbumin are shown in Figure 2. Alpha lactalbumin, beta lactalbumin B, and beta lactalbumin A all exhibit characteristic absorption at 200 nm and 279 nm, respectively.

3.3 Continuous injection superposition spectrum of mixed standard solution of alpha lactalbumin and beta lactalbumin

Figure 3 Continuous injection superposition spectrum of alpha lactalbumin and beta lactalbumin mixed standard solution (100 μ g/mL, n=4) (click to view large image)

The mixed standard solution of alpha lactalbumin and beta lactalbumin (100 μ g/mL) was injected four times in a row, and the RSD of retention time and peak area were both less than 0.3%. The method has good reproducibility, and the separation spectrum and data are shown in Figure 3.

3.4 Overlay spectrum of calibration curves for alpha lactalbumin and beta lactalbumin standard solutions

Figure 4 Stacked spectrum of calibration curves for alpha lactalbumin and beta lactalbumin standard solutions (0.00/40.0/80.0/120/160/200 μ g/mL) (Click to view larger image)

The content of alpha lactalbumin and beta lactoglobulin in raw milk and pasteurized milk is usually high, so the linear ranges recommended by T/TDSTIA 002-2021 and T/TDSTIA 007-2019 group standards were selected. As shown in Figure 4, the linear range of α - lactalbumin standard solution is 0.00-200 μ g/mL, with a linear correlation coefficient r²≥ 0.9997, linear range of β - lactoglobulin standard solution 0.00-200 μ g/mL, linear correlation coefficient r²≥ 0.9991, with good linearity. Among them, β - lactoglobulin B and β - lactoglobulin A were quantified using compound groups.

Overlay spectrum of 3.5 α - lactalbumin and β - lactoglobulin standard solution (40.0 μ g/mL) with blank solution

Figure 5: Overlay spectrum of alpha lactalbumin and beta lactalbumin standard solution (40.0 μ g/mL) with blank solution (click to view larger image)

When the injection volume is 10 uL, the peak heights of alpha lactalbumin and beta lactalbumin (40.0 μ g/mL) are taken as S, and the average value of matrix noise from 22.5-24.0 min is selected as N. The signal-to-noise ratios (S/N) of alpha lactalbumin, beta lactalbumin B, and beta lactalbumin A are 523.3, 139.9, and 133.6, respectively, as shown in Figure 5. Therefore, with the optimization of injection volume, the sensitivity of the alpha lactalbumin and beta lactalbumin determination methods can also meet the requirement of 1.00 μ g/mL in NY/T 4630-2025.

3.6 Overlay spectrum of mixed standard solution of alpha lactalbumin and beta lactoglobulin and pasteurized milk sample solution

Figure 6: Overlay spectrum of a mixed standard solution of alpha lactalbumin and beta lactoglobulin with a pasteurized milk sample solution (click to view larger image)

In addition to whey protein, cow's milk also contains a large amount of casein and other proteins, which may interfere with the determination of alpha lactalbumin and beta lactalbumin. After pre-treatment operations such as pH adjustment and high-speed centrifugation, the undenatured α - lactalbumin and β - lactoglobulin in the pasteurized milk sample were retained in the supernatant. The separated and retained peak shapes were good, the baseline was stable, and there was no interference when the peak appeared, as shown in Figure 6. Using the external standard method for quantification, the alpha lactalbumin content in the pasteurized milk sample was 1033.6 mg/kg and the beta lactoglobulin content was 2949.0 mg/kg, which were basically consistent with the label values.

3.7 Separation spectra and data of pasteurized milk (low-temperature milk), ultra-high temperature sterilized milk (normal temperature milk), and infant formula milk powder sample solutions

Alpha lactalbumin and beta lactalbumin are sensitive to temperature, and high temperatures can damage their chemical structure and biological activity. As shown in Figure 7-9, there are no obvious interference peaks near the target peaks of alpha lactalbumin and beta lactoglobulin in pasteurized milk (low-temperature milk), which can be accurately determined and quantified. Raw milk, pasteurized milk and other samples are suitable for the first method of NY/T 4630-2025; However, there are significant interference peaks near the target peak of β - lactoglobulin in ultra-high temperature sterilized milk (normal temperature milk) and infant formula milk powder, which can seriously affect qualitative and quantitative results. Alpha lactalbumin is relatively less affected, and samples such as ultra-high temperature sterilized milk and infant formula milk powder are not suitable for the first method of NY/T 4630-2025. It is recommended to use the second method.

3.8 gel permeation chromatogram of α - lactalbumin and β - lactoglobulin standard solution (the second method)

Figure 10 gel permeation chromatogram of α - lactalbumin and β - lactoglobulin standard solution (500 mg/L) (click to view the large picture)

Referring to the article "Determination of α - lactalbumin Content in Milk, Milk Powder, and Whey Powder by Volume Exclusion Chromatography" published by Professor Du Ruyun in the journal "China Dairy Industry" (2022), two MAbPac SEC-1 (300 Å, 5 μ m, 7.8 × 300 mm) volume exclusion chromatography columns were used in series, and 6 M guanidine hydrochloride was used as the mobile phase for isocratic elution. α - lactalbumin and β - lactoglobulin can be effectively separated at 280 nm (as shown in Figure 10), and 60 batches of milk, milk powder, and whey powder were tested to verify the applicability and stability of the method. The relevant spectra and data are detailed in the original text.

The MAbPac SEC-1 chromatographic column specification selected in the article is7.8×300mm， The recommended flow rate is 0.5 mL/min, which can achieve solvent savings while ensuring separation4.0×300Mm specification chromatographic column, recommended flow rate of 0.2 mL/min (can be adjusted appropriately).

Recommended chromatographic consumables for NY/T 4630-2025

References