The Analysis of Lactose in Milk and Cheese Products by HPLC With RI Detection
The Analysis of Lactose in Milk and Cheese Products by HPLC With RI Detection
The Analysis of Lactose in Milk and Cheese Products by HPLC With RI Detection
Roberto Troiano
Francesca Denaro
PerkinElmer, Inc. Italy
Wilhad M. Reuter
PerkinElmer, Inc. Shelton, CT
APPLICATION NOTE
Liquid Chromatography
2
Table Experimental
1. HPLC method parameters.
Hardware/Software
Column: Aminex® HPX-87H, 9 μm 300x7.8-mm column (BioRad, Italy)
For all chromatographic separations, a PerkinElmer Altus HPLC
Mobile Phase: 2% acetonitrile / 98% water (isocratic)
System was used, including the Altus A-10 solvent and sample
Analysis Time: 20 min. module, integrated vacuum
degasser/column oven and RI
Flow Rate: 0.8 mL/min. detector. All instrument
control, analysis and data processing
Pressure: 1100 psi was performed using the Waters®
Empower® 3 Chromatography Data System (CDS) platform.
Oven Temp.: Detection: 65 oC
RI Method Parameters
Injection Volume: 10 μL The HPLC method parameters
are shown in Table 1.
Solvents, Standards and Samples
and filtered into individual 2-mL vials, using 0.22-μm
PTFE filters. All solvents and diluents used were HPLC grade and filtered via
Overall, there was a 10-fold sample dilution during
sample 0.45-μm filters. Water was used for all dilutions.
preparation. 10 μL of each prepared sample was then injected
Standard solutions of lactose, glucose and galactose were prepared.
onto the HPLC.
For all three sugars, a concentration range between 0.005% and
Prior to injection, all calibrants and samples were filtered
through 0.25% was prepared.
0.45-μm filters to remove any small particles.
The samples included mozzarella cheese, ricotta cheese and an easily-digestible low-lactose milk. For each of these, 5 g of
sample was weighed out in a 50-mL volumetric flask. Subsequently, each flask was filled to mark with 7% perchloric acid (HClO
4
Results and Discussion
For lactose, the resulting calibration had a linear fit of R2 = 0.997 and, for both glucose and galactose, the fit was 0.999 (curves
). The acid was used to precipitate the proteins, with
not shown).
the sugars remaining in solution. Each sample flask was then
Figure 1 shows the chromatogram of the 0.005%
(50-ppm) agitated for 10 minutes. The resulting mixtures were brought to
standard solution, with the three analytes separated in
under pH 7 with 0.1 N NaOH (so as to avoid possible hydrolysis of the
eight minutes. The additional 12 minutes were used to
assure lactose), transferred to centrifuge tubes and centrifuged at
that all residual matrix components eluted off of the
column 6000 rpm. 1 mL of supernatant from each flask was collected
between injections.
1.40
1.20
1.00
V M
0.80
0.60
0.40
0.20
0.00
-0.20
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes
Figure 1. Chromatogram of the 0.005% (50 ppm) standard solution of lactose, glucose and galactose; 10-μL inj.
Figures 2 and 3 show the chromatograms of the prepared mozzarella and ricotta cheese, respectively. Only lactose was detected
in each of these cheeses. Adjusting for the 10-fold sample dilution during sample preparation, both cheeses showed significant
amounts of lactose. Though the diluted concentration of 0.275% for ricotta cheese was slightly above the 0.25% high calibration
level, this wasn't considered significant. Subsequently, this could easily be accommodated by using an additional calibration
level.
20.00
Minutes
Figure 2. Chromatogram of the prepared mozzarella sample; 10-μL inj.
Mozzarella
15.00
0.6% Lactose
10.00 V M
5.00
0.00
-5.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
35.00
30.00
Minutes
Figure 3. Chromatogram of the prepared ricotta sample; 10-μL inj.
Ricotta 25.00
2.75% Lactose
V M
20.00
15.00
10.00
5.00
0.00
-5.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
3
Conclusion
Figure 4 shows the chromatogram of the prepared low-lactose
The results demonstrated the effective use of this HPLC
method milk sample. All three sugar analytes were detected. Lactose
for the isocratic separation of lactose, glucose and
galactose and was found at 0.074%. This concentration level was not
the quantitative analysis of lactose in milk and cheeses,
using RI surprising, considering that this particular milk was of the easily-
detection. The method allowed for lactose quantitation
down digestible variety, expected to contain <0.1% lactose. Though
to 0.005% (50 ppm) and the calibration range
encompassed the significant amounts of glucose and galactose were detected, as
highest analyte concentration typically found in such
samples. If they were not the focus of this work, they were not quantitated.
required, additional sensitivity may be obtained by increasing the injection volume, though it is recommended not to go beyond
50 μl, taking care not to overload the column.
160.00
140.00
120.00
Low-lactose Milk 0.074% Lactose 100.00
80.00
60.00
40.00
20.00
0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 Minutes
Figure 4. Chromatogram of the prepared low-lactose milk sample; 10-μL inj.
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