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Analysis of crystallized lactose in milk powder by Fourier-transform infrared spectroscopy combined with two-dimensional correlation infrared spectroscopy
Journal of Molecular Structure 974 (2010) 88–93
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Analysis of crystallized lactose in milk powder by Fourier-transform infrared spectroscopy combined with two-dimensional correlation infrared spectroscopy Yu Lei a, Qun Zhou a, Yan-ling Zhang a, Jian-bo Chen a, Su-qin Sun a,*, Isao Noda b a
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Analysis Center, Tsinghua University, Beijing 100084, PR China b The Procter & Gamble Company, West Chester, OH 45069-7053, USA
a r t i c l e
i n f o
Article history: Received 30 October 2009 Received in revised form 11 December 2009 Accepted 11 December 2009 Available online 22 December 2009 Keywords: Milk powder Infrared spectroscopy (IR) Two-dimensional infrared spectroscopy (2D IR) Crystallized lactose
a b s t r a c t Infrared (IR) spectroscopy is used in combination with two-dimensional (2D) correlation IR spectroscopy to conduct rapid non-destructive quantitative research in milk powder without additional separation steps. The experiments conducted in both FT-IR and 2D FT-IR spectra suggest that characteristic spectroscopic features of milk powder containing different carbohydrate can be detected, and then determine the type of carbohydrate. To predict the approximate content of lactose while the carbohydrate is lactose, different amount of crystallized lactose has been added to the reference milk powder. The correlation coefficient could be used to determine the content of crystallized lactose in milk powder. The method provides a rapid and convenient means for assessing the quality of milk powder. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Dairy is an inseparable part of our daily lives. Milk powder has a far longer shelf life than liquid milk and is widely used because of the reduced transport and storage costs. It is frequently used in the manufacture of infant formulas and various dairy foods. However, some complicated and time consuming techniques are still used in the quality control of milk powder. For example, China still typically uses the semi-micro Kjeldahl nitrogen determination method that has been in use since 1985 to analyze the protein from milk powder. Likewise, the Rose–Gettlieb method (1985) is used to test the liquid milk. As for the sugar analysis, JH Lave and Egnon method (1985) is still applied [1]. Fortunately, because of the introduction of some new techniques, such as HPLC, JH Lave and Egnon type methods are nowadays employed as supplementary techniques. Obviously, these out-of-dated methods fail to meet some of the current detection challenges encountered in the food quality assurance. For example, milk adulteration poses a major threat to China’s nutritional needs. There is usually a certain amount of amorphous lactose in the milk powder, but the lactose becomes crystallized when stored for a long period of time. Some manufacturers are directly adding some crystallized lactose to the milk powder to save the cost. In the traditional method, we must convert the lactose * Corresponding author. Tel.: +86 10 62781692; fax: +86 10 62782485. E-mail address: [email protected] (S.-q. Sun). 0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.12.030
first and then determine the nature of the adulterant. The process unfortunately is cumbersome and complex. Based on the importance of the quality control of milk powder and there is only a limited amount of studies on this subject at home and abroad [2–6], we want to create a rapid and effective detection method to better control the quality of milk powder products. Fourier-transform infrared spectroscopy (FT-IR) is one of the most widely used methods to identify chemical compounds and elucidate chemical structures. It is adopted by the food industry to test the quality of food products based on the fingerprinting features of IR profiles. Currently, this technology is used in many fields, such as food science, traditional Chinese medicine, chemical industry applications, and so on [7–12]. By interpreting the characteristic peaks of chemical functional groups, the representative chemical constituents (e.g., fat, protein, and carbohydrates) of the milk powder could be revealed. Two-dimensional (2D) correlation analysis [13] provides a convenient way to monitor the relationship among different functional groups in molecules. The so-called generalized 2D correlation spectroscopy has become a surprisingly powerful tool for the detailed analysis of spectral data of various complex systems [14]. In this paper, IR and 2D-IR spectroscopy are used to study the type of carbohydrate and the crystallized lactose content of milk powder without additional separation steps. To predict the approximate lactose content in commercial milk powder, different amounts of crystallized lactose have been added to a milk powder without crystallized lactose to obtain the reference spectra. If any
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
crystallized lactose is added to milk powder, we should be able to rapidly determine its content by using the reference spectra. 2. Experiment 2.1. Apparatus The measurements were carried out by using a FT-IR spectrometer (Spectrum GX, Perkin–Elmer Ltd., England), equipped with a DTGS detector. IR spectra were recorded at 32 scans in 4000– 400 cm1 range with a resolution of 4 cm1. A programmable temperature controller (Model 50-886, Love Control Corporation) was used to perform the thermal perturbation.
89
main components, fat, protein and lactose, all have their own characteristic peaks. IR peaks at 2924, 2853, 1746 and 1159 cm1 arise from the fat in the milk powder. As the fat contents decreased, the intensity of characteristic peak of C=O bond in fat at 1746 cm1 and the intensity of 1161 cm1 which represents C–O vibration in fat are noticeably decreasing. So we can determine the fat content of milk powder through the intensity of the peak 1746 and 1161 cm1. Two broad peaks with middle intensity around 1658 and 1544 cm1 stand for the vibrations of amide I and II of protein. The area between 800 and 1250 cm1 embodies characteristic peaks of various C–O vibrations in carbohydrates. We can determine the type of crystallized sugar added into the milk powder by the characteristic peak in the area. The main absorptive peaks of FT-IR spectra were listed in Table 2.
2.2. Materials 3.2. FT-IR spectra of milk powder with different amount of lactose Various kinds of milk powder products were bought from the local market in Beijing, China (Table 1). 2.3. Methods (1) One g crystallized lactose was weighed accurately and added to 9 g reference milk powder. Mixed them evenly with ultrasonic vibration, and made a sample with 10% crystallized lactose. Made other samples with 20–90% crystallized lactose respectively applying the same method. (2) Each milk powder sample was ground with KBr powder and then pressed into a tablet. Subsequently, the IR spectrum was collected. (3) The tablet was put into the sample pool with programmable temperature controller and then collected the dynamic spectra at different temperatures from 50 to 120 °C at interval of 10 °C. (4) Using the analysis software of the Perkin–Elmer company, compare the similarity between the two infrared spectroscopy (or between a spectrum and a series of spectra). The correlation coefficient could be obtained use following formula
P
wi Ai Bi k¼ P P ð wi Ai Ai Þ1=2 ð wi Bi Bi Þ1=2
ð1Þ
where k is the correlation coefficient, Ai and Bi are the absorbance values in spectra A and B at frequency i, and wi is a weighting that depends on the filters selected. Each spectrum was processed via baseline-correction first. 2DIR correlation spectra were obtained by treating the series of temperature-dependent dynamic spectra with homemade 2D-IR correlation analysis software.
Currently, most infant formula milk powder sold in the Chinese market is added with crystallized lactose. Thus, we took a lactose free milk powder and added known amounts of crystallized lactose to obtain a set of reference samples. By taking advantage of the regularity of the composition-dependent spectral features, one can estimate the content of the crystallized lactose in an unknown milk powder sample. The FT-IR spectra of milk powder of the different amount of lactose are shown in Fig. 2. When the content of lactose is getting higher, there is a small shoulder peak at 3528 cm1. The peaks of fat at 2924, 2853, 1746, and 1159 cm1 and of protein at 1658 and 1544 cm1 are weakening, while the peaks of lactose are gradually getting stronger. In the range from 1150 to 1030 cm1 of the above spectra, there are some C–O peaks groups of the lactose, the peaks are getting stronger and peak shapes are getting similar to pure lactose. In addition, the peaks of the lactose skeleton at 899, 875 and 778 cm1 are getting stronger as more lactose is added. 3.3. The second derivative FT-IR of milk powder with different amount of lactose Generally, the second derivative IR spectra can enhance the resolution obviously and amplify even tiny difference in ordinary IR spectra. By using the technique, some overlapped absorption peaks can be distinguished. Fig. 3 shows the second derivative FT-IR spectra for the different amount of lactose in the milk powder. As the content of the crystallized lactose increases, the intensity of characteristic peak for lactose at 3528 cm1 is noticeably increasing, and the intensity of characteristic peak for fat is decreasing. Meanwhile, in the fingerprint region from 800 to 1000 cm1, peak intensities at 917, 897 and 973 cm1 are increasing, which stand for carbohydrate ring of the crystallized lactose.
3. Results and discussion 3.1. FT-IR spectra of milk powder with different amount of fat Fig. 1 shows the FT-IR spectra of whole milk powder (WMP), low-fat milk powder (LFMP) and skim milk powder (SMP). Three Table 1 The contents of main components in various milk powder on the products label. Milk powder
Carbohydrates (%)
Milk protein (%)
Milk fat (%)
Manufacturer
A B C D E
57.7 54 57.0 54.4 51
11.0 15.0 10.8 17.7 21.6
27.1 23 27.0 18 19
QUECAO SHENGYUAN MEISUJIAER QUECAO QUECAO
4. Milk powder of different manufacturers with unknown lactose contents The infant milk powder samples with unknown types of carbohydrates were selected from the supermarket of Beijing. We hope to determine the type of carbohydrate, and then estimate the lactose content of the milk powder according to the above reference spectroscopy if the additive is lactose. 4.1. FT-IR spectra of milk powder from different manufactures Fig. 4 shows the conventional (1D) IR spectrum of infant milk powder. It is clear that they have distinct fingerprint features, particularly in the range of 1000–1200 cm1 and 800–1000 cm1. In
90
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
2925
2854
1747 1658 1457 1541
1037 780 876 705 894
1244 1035
a Absorbance
1072
1072 1144
2925 2854
1658 1440 1544 1260
1747
779 876 705 898 1039
b
1071 1663 1405
2925
1544
702 1248
782 892
c 4000.0
3000
2000
1500
1000
400.0
wavenumber(cm-1) Fig. 1. FT-IR spectra of milk powder with different amount of fat (a) WMP, (b) LFMP and (c) SMP.
Table 2 The main absorption peak and the attribution of milk powder. Wavenumber (cm1)
Group
Vibration mode
Attribution
2925–2927 2854–2855 1747 1660–1650 1541–1547 1241–1251 1160 1150–1030 800–1000 722 700
Methylene (–CH2) Methylene (–CH2) Carbonyl (C=O) Carbonyl (C=O) N–H mainly C–N mainly C–O C–O C–C C–O–C Carbohydrate ring –(CH2)n– Amide (N–H) mainly
Stretching antisymmetrically Stretching symmetrically Stretching Stretching Bending inside Stretching Stretching Stretching Ring vibration Swinging inside Bending outside
Fat mainly Fat mainly Fatl Protein (amide I) Protein (amide II) Protein (amide III) Fat mainly Carbohydrate Carbohydrate Oil Protein
2925
1660 1464 1747 2854 2854
a
1747 1659 1458 1545
2925
b 2854
1747 1660 1459 1540
Absorbance
2925 2855 3528
c
1660 1747
2926
2928
1659 1747
2855
e 3529
2901 2934
f 4000.0
3000
1163 1094
706 891
1165 1094 549
897
1095 1167
714
1094 1167 1457 1261
1540
d 3528
1545
898 875 898 778
1094 1168 1437 1261 899 778 876 1094 1436 1168 875 778 1261 899
551
551
551
1659
2000
1500
1000
400.0
Wavenumber(cm-1) Fig. 2. FT-IR spectra of milk powder with different amount of lactose (a) 0.0%, (b) 10%, (c) 20%, (d) 30%, (e) 40% and (f) 100%.
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
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Fig. 3. The second derivative FT-IR of milk powder with different amount of lactose (a) 0.0%, (b) 10%, (c) 20%, (d) 30%, (e) 40% and (f) 100%.
the range of 1000–1200 cm1, there are three peaks at 1116, 1071 and 1036 cm1 for the milk powder of the A, B and C manufactures. Likewise, in the range of 800–1000 cm1, there are three peaks at 896, 875 and 779 cm1. Comparison of the IR spectra of the milk powder of the A, B, C manufacture and crystallized lactose reveals that the peaks positions and shapes of the main specific bands in the spectrum are quite similar to each other, so it can be determined that they all contain crystallized lactose. But in the range of 1000–1200 cm1, there are two peaks at 1069 and 1050 cm1 for the milk powder of the D and E manufactures. Similarly, in the range of 800–1000 cm1, there are two peaks at 909 and
868 cm1. The IR spectra of them are different from the crystallized lactose, so it can be determined that they do not contain crystallized lactose. 4.2. The second derivative FT-IR of milk powder from different manufactures The second derivative IR spectra of milk powder of the A, B, C, D, E manufacture and the crystallized lactose are shown in Fig. 5. Many similarities and differences invisible in FT-IR spectra become clearer; especially in the range from 3000 to 3600 cm1 and from
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Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
Fig. 4. FT-IR spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose (F).
960 to 1200 cm1. In the range of 3000–3600 cm1, there are three similar peaks at 3525, 3381 and 3339 cm1 for the milk powder of the A, B, C manufacture and the crystallized lactose. Likewise, in the range of 960–1200 cm1, there are eleven similar peaks at 1168, 1144, 1134, 1117, 1095, 1083, 1057, 1017, 1018, 1003 and 987 cm1. But the peaks positions are different between the D, E manufacture and the crystallized lactose in the range from 3000 to 3600 cm1 and from 960 to 1200 cm1. Hence, it can be identified easily by those enlarged fingerprint characters in the second derivative IR spectra which the milk powder of the A, B, C manufacture contain the crystallized lactose but the D, E manufacture not. 4.3. 2D FTIR FT-IR of milk powder from different manufactures It has been shown that 2D-IR spectra can enhance the spectral resolution and obtain much new information which cannot be acquired from IR spectra and second derivative spectra. Therefore, it
can provide visual and legible spectra for discriminating the similar complicated samples. The type of carbohydrate among the different manufactures can be described further through the 2D-IR synchronous spectra (Fig. 6). The correlation peaks in synchronous spectrum represent a related IR vibrations spectrum, the auto-peaks on the diagonal line show the self-correlativity and susceptibility of some normal vibration of functional group with the increasing temperature, and the cross peaks located at the off-diagonal position reveal the relativity of intensity variations of auto-peak will be. We can see clearly some similarities and differences among the milk powder of the A, B, C, D and E manufacture in the 2D-IR spectra. For example, there is only one distinct auto-peak at 1136 cm1 in synchronous spectrum of the milk powder of the A, B, C manufacture and the crystallized lactose within 1000–1200 cm1, while in the milk powder of the D, E manufacture there are two auto-peak situated at 1029 and 1058 cm1, and they have self-correlativity in
Fig. 5. The second derivative FT-IR spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose.
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
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Fig. 6. 2D correlation spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose from 1000 to 1200 cm.1
Table 3 The correlation coefficient between unknown milk powder and the milk powder with different amount crystallized lactose contents. Number
Milk powder
10% Lactose
20% Lactose
30% Lactose
40% Lactose
Lactose
A B C
0.9717 0.9803 0.9733
0.9857 0.9929 0.9853
0.9799 0.9928 0.9867
0.9538 0.9514 0.9635
0.8152 0.8015 0.8067
0.1800 0.1711 0.1925
the increasing temperature process. The spectral differences are the objective embodiment of the differences of chemical constituents in the samples, so we know the carbohydrates in the milk powder of the A, B and C manufacture are different from the D and E manufacture. 4.4. The content of crystallized lactose estimated by correlation coefficient So it can be determined that the milk powder of the A, B, C manufactures containing crystallized lactose. By comparing with the reference spectra, the contents of crystallized lactose in milk powder could be estimated by using the correlation coefficient. The results are shown in Table 3. In Table 3, IR spectrum of the milk powder from the manufacture A compared with the spectrum of milk powder with 10% crystallized lactose gives the highest correlation coefficient of 0.9857. Thus, the crystallized lactose content of the milk powder sample from the manufacture A is determined to be about 10%. In a similar manner, when the milk powder of the manufacture B is compared with the milk powder of 10% and 20% crystallized lactose content, correlation coefficients are both high at 0.9929, 0.9928. So it was determined that the crystallized lactose content of milk powder from the B manufacture must be about 10–20%. The milk powder of the manufacture C compares well with the milk powder of 20% crystallized lactose content; correlation coefficient is the highest at 0.9867. So the crystallized lactose content of milk powder from the C manufacture is about 20%.
5. Conclusions Results from IR spectroscopy and the second derivative FT-IR combined with 2D IR correlation spectroscopy successfully exhibit protein, fat and carbohydrates in different milk powder. By summarizing the regularity of the IR spectra, the second derivative spectra, and 2D IR spectra, the types of the additives in unknown milk powder are determined. We further confirm the contents of crystallized lactose in unknown milk powder through the correlation coefficient. The label on the milk powder package usually shows rough carbohydrate contents. Yet, it merely gives customers more details on the exact sugar contents. Therefore, most customers have hard time to understand the exact additives that are added to the milk powder. They are not sure which milk powder would be the best to meet their nutritional needs either. Fortunately, IR spectroscopy is able to correctly determine the contents of the additives used in the milk powder and produces the best results for correct classification. Using the IR spectroscopy, we can conduct rapid non-destructive quantitative analysis to test the quality of milk powder effectively. References [1] National Standards of the People’s Republic of China. Analytical methods for milk powder, GB 5413-85. [2] Z. Qin, C.H. Xu, Q. Zhou, et al., Chin. J. Anal. Chem. 32 (9) (2004) 1156. [3] Y.E. Deng, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 25 (12) (2005) 1972. [4] Y.E. Deng, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 26 (4) (2006) 636. [5] Y.E. Deng, S.Q. Sun, L.Y. Niu, et al., Acta Nutr. Sin. 29 (1) (2007) 91. [6] Q. Zhou, Su-qin Sun, Lu Yu, et al., J. Mol. Struct. 799 (1–3) (2006) 77. [7] H.X. Liu, S.Q. Sun, G.H. Lv, et al., Spectrochim. Acta Part A. 64 (2) (2006) 321. [8] C.H. Xu, S.Q. Sun, C.Q. Guo, Q. Zhou, et al., Vib. Spectrosc. 41 (1) (2006) 118. [9] Y.M. Li, S.Q. Sun, Q. Zhou, et al., Spectrochim. Acta Part A. 63 (3) (2006) 565. [10] L. Yu, S.Q. Sun, K.F. Fan, Q. Zhou, I. Noda, Spectrochim. Acta Part A. 62 (1–3) (2005) 22. [11] M.H. Liu, X.G. Zhang, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 25 (6) (2005) 878. [12] M.J. Liu, Z. Wang, S.Q. Sun, Q.Y. Wu, Spectrosc. Spect. Anal. 25 (5) (2005) 688. [13] I. Noda, J. Am. Chem. Soc. 111 (21) (1989) 8116. [14] I. Noda, Appl. Spectrosc. 47 (9) (1993) 1329.
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Analysis of crystallized lactose in milk powder by Fourier-transform infrared spectroscopy combined with two-dimensional correlation infrared spectroscopy Yu Lei a, Qun Zhou a, Yan-ling Zhang a, Jian-bo Chen a, Su-qin Sun a,*, Isao Noda b a
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Analysis Center, Tsinghua University, Beijing 100084, PR China b The Procter & Gamble Company, West Chester, OH 45069-7053, USA
a r t i c l e
i n f o
Article history: Received 30 October 2009 Received in revised form 11 December 2009 Accepted 11 December 2009 Available online 22 December 2009 Keywords: Milk powder Infrared spectroscopy (IR) Two-dimensional infrared spectroscopy (2D IR) Crystallized lactose
a b s t r a c t Infrared (IR) spectroscopy is used in combination with two-dimensional (2D) correlation IR spectroscopy to conduct rapid non-destructive quantitative research in milk powder without additional separation steps. The experiments conducted in both FT-IR and 2D FT-IR spectra suggest that characteristic spectroscopic features of milk powder containing different carbohydrate can be detected, and then determine the type of carbohydrate. To predict the approximate content of lactose while the carbohydrate is lactose, different amount of crystallized lactose has been added to the reference milk powder. The correlation coefficient could be used to determine the content of crystallized lactose in milk powder. The method provides a rapid and convenient means for assessing the quality of milk powder. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Dairy is an inseparable part of our daily lives. Milk powder has a far longer shelf life than liquid milk and is widely used because of the reduced transport and storage costs. It is frequently used in the manufacture of infant formulas and various dairy foods. However, some complicated and time consuming techniques are still used in the quality control of milk powder. For example, China still typically uses the semi-micro Kjeldahl nitrogen determination method that has been in use since 1985 to analyze the protein from milk powder. Likewise, the Rose–Gettlieb method (1985) is used to test the liquid milk. As for the sugar analysis, JH Lave and Egnon method (1985) is still applied [1]. Fortunately, because of the introduction of some new techniques, such as HPLC, JH Lave and Egnon type methods are nowadays employed as supplementary techniques. Obviously, these out-of-dated methods fail to meet some of the current detection challenges encountered in the food quality assurance. For example, milk adulteration poses a major threat to China’s nutritional needs. There is usually a certain amount of amorphous lactose in the milk powder, but the lactose becomes crystallized when stored for a long period of time. Some manufacturers are directly adding some crystallized lactose to the milk powder to save the cost. In the traditional method, we must convert the lactose * Corresponding author. Tel.: +86 10 62781692; fax: +86 10 62782485. E-mail address: [email protected] (S.-q. Sun). 0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.12.030
first and then determine the nature of the adulterant. The process unfortunately is cumbersome and complex. Based on the importance of the quality control of milk powder and there is only a limited amount of studies on this subject at home and abroad [2–6], we want to create a rapid and effective detection method to better control the quality of milk powder products. Fourier-transform infrared spectroscopy (FT-IR) is one of the most widely used methods to identify chemical compounds and elucidate chemical structures. It is adopted by the food industry to test the quality of food products based on the fingerprinting features of IR profiles. Currently, this technology is used in many fields, such as food science, traditional Chinese medicine, chemical industry applications, and so on [7–12]. By interpreting the characteristic peaks of chemical functional groups, the representative chemical constituents (e.g., fat, protein, and carbohydrates) of the milk powder could be revealed. Two-dimensional (2D) correlation analysis [13] provides a convenient way to monitor the relationship among different functional groups in molecules. The so-called generalized 2D correlation spectroscopy has become a surprisingly powerful tool for the detailed analysis of spectral data of various complex systems [14]. In this paper, IR and 2D-IR spectroscopy are used to study the type of carbohydrate and the crystallized lactose content of milk powder without additional separation steps. To predict the approximate lactose content in commercial milk powder, different amounts of crystallized lactose have been added to a milk powder without crystallized lactose to obtain the reference spectra. If any
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
crystallized lactose is added to milk powder, we should be able to rapidly determine its content by using the reference spectra. 2. Experiment 2.1. Apparatus The measurements were carried out by using a FT-IR spectrometer (Spectrum GX, Perkin–Elmer Ltd., England), equipped with a DTGS detector. IR spectra were recorded at 32 scans in 4000– 400 cm1 range with a resolution of 4 cm1. A programmable temperature controller (Model 50-886, Love Control Corporation) was used to perform the thermal perturbation.
89
main components, fat, protein and lactose, all have their own characteristic peaks. IR peaks at 2924, 2853, 1746 and 1159 cm1 arise from the fat in the milk powder. As the fat contents decreased, the intensity of characteristic peak of C=O bond in fat at 1746 cm1 and the intensity of 1161 cm1 which represents C–O vibration in fat are noticeably decreasing. So we can determine the fat content of milk powder through the intensity of the peak 1746 and 1161 cm1. Two broad peaks with middle intensity around 1658 and 1544 cm1 stand for the vibrations of amide I and II of protein. The area between 800 and 1250 cm1 embodies characteristic peaks of various C–O vibrations in carbohydrates. We can determine the type of crystallized sugar added into the milk powder by the characteristic peak in the area. The main absorptive peaks of FT-IR spectra were listed in Table 2.
2.2. Materials 3.2. FT-IR spectra of milk powder with different amount of lactose Various kinds of milk powder products were bought from the local market in Beijing, China (Table 1). 2.3. Methods (1) One g crystallized lactose was weighed accurately and added to 9 g reference milk powder. Mixed them evenly with ultrasonic vibration, and made a sample with 10% crystallized lactose. Made other samples with 20–90% crystallized lactose respectively applying the same method. (2) Each milk powder sample was ground with KBr powder and then pressed into a tablet. Subsequently, the IR spectrum was collected. (3) The tablet was put into the sample pool with programmable temperature controller and then collected the dynamic spectra at different temperatures from 50 to 120 °C at interval of 10 °C. (4) Using the analysis software of the Perkin–Elmer company, compare the similarity between the two infrared spectroscopy (or between a spectrum and a series of spectra). The correlation coefficient could be obtained use following formula
P
wi Ai Bi k¼ P P ð wi Ai Ai Þ1=2 ð wi Bi Bi Þ1=2
ð1Þ
where k is the correlation coefficient, Ai and Bi are the absorbance values in spectra A and B at frequency i, and wi is a weighting that depends on the filters selected. Each spectrum was processed via baseline-correction first. 2DIR correlation spectra were obtained by treating the series of temperature-dependent dynamic spectra with homemade 2D-IR correlation analysis software.
Currently, most infant formula milk powder sold in the Chinese market is added with crystallized lactose. Thus, we took a lactose free milk powder and added known amounts of crystallized lactose to obtain a set of reference samples. By taking advantage of the regularity of the composition-dependent spectral features, one can estimate the content of the crystallized lactose in an unknown milk powder sample. The FT-IR spectra of milk powder of the different amount of lactose are shown in Fig. 2. When the content of lactose is getting higher, there is a small shoulder peak at 3528 cm1. The peaks of fat at 2924, 2853, 1746, and 1159 cm1 and of protein at 1658 and 1544 cm1 are weakening, while the peaks of lactose are gradually getting stronger. In the range from 1150 to 1030 cm1 of the above spectra, there are some C–O peaks groups of the lactose, the peaks are getting stronger and peak shapes are getting similar to pure lactose. In addition, the peaks of the lactose skeleton at 899, 875 and 778 cm1 are getting stronger as more lactose is added. 3.3. The second derivative FT-IR of milk powder with different amount of lactose Generally, the second derivative IR spectra can enhance the resolution obviously and amplify even tiny difference in ordinary IR spectra. By using the technique, some overlapped absorption peaks can be distinguished. Fig. 3 shows the second derivative FT-IR spectra for the different amount of lactose in the milk powder. As the content of the crystallized lactose increases, the intensity of characteristic peak for lactose at 3528 cm1 is noticeably increasing, and the intensity of characteristic peak for fat is decreasing. Meanwhile, in the fingerprint region from 800 to 1000 cm1, peak intensities at 917, 897 and 973 cm1 are increasing, which stand for carbohydrate ring of the crystallized lactose.
3. Results and discussion 3.1. FT-IR spectra of milk powder with different amount of fat Fig. 1 shows the FT-IR spectra of whole milk powder (WMP), low-fat milk powder (LFMP) and skim milk powder (SMP). Three Table 1 The contents of main components in various milk powder on the products label. Milk powder
Carbohydrates (%)
Milk protein (%)
Milk fat (%)
Manufacturer
A B C D E
57.7 54 57.0 54.4 51
11.0 15.0 10.8 17.7 21.6
27.1 23 27.0 18 19
QUECAO SHENGYUAN MEISUJIAER QUECAO QUECAO
4. Milk powder of different manufacturers with unknown lactose contents The infant milk powder samples with unknown types of carbohydrates were selected from the supermarket of Beijing. We hope to determine the type of carbohydrate, and then estimate the lactose content of the milk powder according to the above reference spectroscopy if the additive is lactose. 4.1. FT-IR spectra of milk powder from different manufactures Fig. 4 shows the conventional (1D) IR spectrum of infant milk powder. It is clear that they have distinct fingerprint features, particularly in the range of 1000–1200 cm1 and 800–1000 cm1. In
90
Y. Lei et al. / Journal of Molecular Structure 974 (2010) 88–93
2925
2854
1747 1658 1457 1541
1037 780 876 705 894
1244 1035
a Absorbance
1072
1072 1144
2925 2854
1658 1440 1544 1260
1747
779 876 705 898 1039
b
1071 1663 1405
2925
1544
702 1248
782 892
c 4000.0
3000
2000
1500
1000
400.0
wavenumber(cm-1) Fig. 1. FT-IR spectra of milk powder with different amount of fat (a) WMP, (b) LFMP and (c) SMP.
Table 2 The main absorption peak and the attribution of milk powder. Wavenumber (cm1)
Group
Vibration mode
Attribution
2925–2927 2854–2855 1747 1660–1650 1541–1547 1241–1251 1160 1150–1030 800–1000 722 700
Methylene (–CH2) Methylene (–CH2) Carbonyl (C=O) Carbonyl (C=O) N–H mainly C–N mainly C–O C–O C–C C–O–C Carbohydrate ring –(CH2)n– Amide (N–H) mainly
Stretching antisymmetrically Stretching symmetrically Stretching Stretching Bending inside Stretching Stretching Stretching Ring vibration Swinging inside Bending outside
Fat mainly Fat mainly Fatl Protein (amide I) Protein (amide II) Protein (amide III) Fat mainly Carbohydrate Carbohydrate Oil Protein
2925
1660 1464 1747 2854 2854
a
1747 1659 1458 1545
2925
b 2854
1747 1660 1459 1540
Absorbance
2925 2855 3528
c
1660 1747
2926
2928
1659 1747
2855
e 3529
2901 2934
f 4000.0
3000
1163 1094
706 891
1165 1094 549
897
1095 1167
714
1094 1167 1457 1261
1540
d 3528
1545
898 875 898 778
1094 1168 1437 1261 899 778 876 1094 1436 1168 875 778 1261 899
551
551
551
1659
2000
1500
1000
400.0
Wavenumber(cm-1) Fig. 2. FT-IR spectra of milk powder with different amount of lactose (a) 0.0%, (b) 10%, (c) 20%, (d) 30%, (e) 40% and (f) 100%.
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Fig. 3. The second derivative FT-IR of milk powder with different amount of lactose (a) 0.0%, (b) 10%, (c) 20%, (d) 30%, (e) 40% and (f) 100%.
the range of 1000–1200 cm1, there are three peaks at 1116, 1071 and 1036 cm1 for the milk powder of the A, B and C manufactures. Likewise, in the range of 800–1000 cm1, there are three peaks at 896, 875 and 779 cm1. Comparison of the IR spectra of the milk powder of the A, B, C manufacture and crystallized lactose reveals that the peaks positions and shapes of the main specific bands in the spectrum are quite similar to each other, so it can be determined that they all contain crystallized lactose. But in the range of 1000–1200 cm1, there are two peaks at 1069 and 1050 cm1 for the milk powder of the D and E manufactures. Similarly, in the range of 800–1000 cm1, there are two peaks at 909 and
868 cm1. The IR spectra of them are different from the crystallized lactose, so it can be determined that they do not contain crystallized lactose. 4.2. The second derivative FT-IR of milk powder from different manufactures The second derivative IR spectra of milk powder of the A, B, C, D, E manufacture and the crystallized lactose are shown in Fig. 5. Many similarities and differences invisible in FT-IR spectra become clearer; especially in the range from 3000 to 3600 cm1 and from
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Fig. 4. FT-IR spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose (F).
960 to 1200 cm1. In the range of 3000–3600 cm1, there are three similar peaks at 3525, 3381 and 3339 cm1 for the milk powder of the A, B, C manufacture and the crystallized lactose. Likewise, in the range of 960–1200 cm1, there are eleven similar peaks at 1168, 1144, 1134, 1117, 1095, 1083, 1057, 1017, 1018, 1003 and 987 cm1. But the peaks positions are different between the D, E manufacture and the crystallized lactose in the range from 3000 to 3600 cm1 and from 960 to 1200 cm1. Hence, it can be identified easily by those enlarged fingerprint characters in the second derivative IR spectra which the milk powder of the A, B, C manufacture contain the crystallized lactose but the D, E manufacture not. 4.3. 2D FTIR FT-IR of milk powder from different manufactures It has been shown that 2D-IR spectra can enhance the spectral resolution and obtain much new information which cannot be acquired from IR spectra and second derivative spectra. Therefore, it
can provide visual and legible spectra for discriminating the similar complicated samples. The type of carbohydrate among the different manufactures can be described further through the 2D-IR synchronous spectra (Fig. 6). The correlation peaks in synchronous spectrum represent a related IR vibrations spectrum, the auto-peaks on the diagonal line show the self-correlativity and susceptibility of some normal vibration of functional group with the increasing temperature, and the cross peaks located at the off-diagonal position reveal the relativity of intensity variations of auto-peak will be. We can see clearly some similarities and differences among the milk powder of the A, B, C, D and E manufacture in the 2D-IR spectra. For example, there is only one distinct auto-peak at 1136 cm1 in synchronous spectrum of the milk powder of the A, B, C manufacture and the crystallized lactose within 1000–1200 cm1, while in the milk powder of the D, E manufacture there are two auto-peak situated at 1029 and 1058 cm1, and they have self-correlativity in
Fig. 5. The second derivative FT-IR spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose.
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Fig. 6. 2D correlation spectra of milk powder on milk powder of the A, B, C, D, E manufacture and the crystallized lactose from 1000 to 1200 cm.1
Table 3 The correlation coefficient between unknown milk powder and the milk powder with different amount crystallized lactose contents. Number
Milk powder
10% Lactose
20% Lactose
30% Lactose
40% Lactose
Lactose
A B C
0.9717 0.9803 0.9733
0.9857 0.9929 0.9853
0.9799 0.9928 0.9867
0.9538 0.9514 0.9635
0.8152 0.8015 0.8067
0.1800 0.1711 0.1925
the increasing temperature process. The spectral differences are the objective embodiment of the differences of chemical constituents in the samples, so we know the carbohydrates in the milk powder of the A, B and C manufacture are different from the D and E manufacture. 4.4. The content of crystallized lactose estimated by correlation coefficient So it can be determined that the milk powder of the A, B, C manufactures containing crystallized lactose. By comparing with the reference spectra, the contents of crystallized lactose in milk powder could be estimated by using the correlation coefficient. The results are shown in Table 3. In Table 3, IR spectrum of the milk powder from the manufacture A compared with the spectrum of milk powder with 10% crystallized lactose gives the highest correlation coefficient of 0.9857. Thus, the crystallized lactose content of the milk powder sample from the manufacture A is determined to be about 10%. In a similar manner, when the milk powder of the manufacture B is compared with the milk powder of 10% and 20% crystallized lactose content, correlation coefficients are both high at 0.9929, 0.9928. So it was determined that the crystallized lactose content of milk powder from the B manufacture must be about 10–20%. The milk powder of the manufacture C compares well with the milk powder of 20% crystallized lactose content; correlation coefficient is the highest at 0.9867. So the crystallized lactose content of milk powder from the C manufacture is about 20%.
5. Conclusions Results from IR spectroscopy and the second derivative FT-IR combined with 2D IR correlation spectroscopy successfully exhibit protein, fat and carbohydrates in different milk powder. By summarizing the regularity of the IR spectra, the second derivative spectra, and 2D IR spectra, the types of the additives in unknown milk powder are determined. We further confirm the contents of crystallized lactose in unknown milk powder through the correlation coefficient. The label on the milk powder package usually shows rough carbohydrate contents. Yet, it merely gives customers more details on the exact sugar contents. Therefore, most customers have hard time to understand the exact additives that are added to the milk powder. They are not sure which milk powder would be the best to meet their nutritional needs either. Fortunately, IR spectroscopy is able to correctly determine the contents of the additives used in the milk powder and produces the best results for correct classification. Using the IR spectroscopy, we can conduct rapid non-destructive quantitative analysis to test the quality of milk powder effectively. References [1] National Standards of the People’s Republic of China. Analytical methods for milk powder, GB 5413-85. [2] Z. Qin, C.H. Xu, Q. Zhou, et al., Chin. J. Anal. Chem. 32 (9) (2004) 1156. [3] Y.E. Deng, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 25 (12) (2005) 1972. [4] Y.E. Deng, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 26 (4) (2006) 636. [5] Y.E. Deng, S.Q. Sun, L.Y. Niu, et al., Acta Nutr. Sin. 29 (1) (2007) 91. [6] Q. Zhou, Su-qin Sun, Lu Yu, et al., J. Mol. Struct. 799 (1–3) (2006) 77. [7] H.X. Liu, S.Q. Sun, G.H. Lv, et al., Spectrochim. Acta Part A. 64 (2) (2006) 321. [8] C.H. Xu, S.Q. Sun, C.Q. Guo, Q. Zhou, et al., Vib. Spectrosc. 41 (1) (2006) 118. [9] Y.M. Li, S.Q. Sun, Q. Zhou, et al., Spectrochim. Acta Part A. 63 (3) (2006) 565. [10] L. Yu, S.Q. Sun, K.F. Fan, Q. Zhou, I. Noda, Spectrochim. Acta Part A. 62 (1–3) (2005) 22. [11] M.H. Liu, X.G. Zhang, Q. Zhou, S.Q. Sun, Spectrosc. Spect. Anal. 25 (6) (2005) 878. [12] M.J. Liu, Z. Wang, S.Q. Sun, Q.Y. Wu, Spectrosc. Spect. Anal. 25 (5) (2005) 688. [13] I. Noda, J. Am. Chem. Soc. 111 (21) (1989) 8116. [14] I. Noda, Appl. Spectrosc. 47 (9) (1993) 1329.