Soft drinks are widely consumed throughout the world. Maintaining and confirming the quality and consistency of a given product from many individual bottling plants is a considerable challenge for central quality control laboratories. The complexity and variety of these samples compound the challenge further. Numbers of samples analyzed each year can reach into the tens of thousands making analysis time important. For example, 10,000 samples analyzed by high performance liquid chromatography (HPLC) at a run time of 16 minutes would require approximately 333, 8-hour working days to complete.
Explore this issueJune/July 2005
This application note shows how an advanced chromatography technique, Ultra Performance LC (UPLC) can reduce run times to under 75 seconds per sample (see Fig. 1), while dealing with a range of analytes in a variety of samples.
The UPLC system consisted of a UPLC binary solvent manager, a UPLC sample manager (5 mL loop) and a UPLC TUV detector. All instruments were controlled and data collected and analyzed using a data chromatography system. Separations were performed on an UPLC bridged ethylsiloxane/silica hybrid (BEH) C18 2.1 X 50 mm, 1.7 mm particle size column at a flow rate of 0.90 mL/ minute.
Column temperature was set to 40°C and injection volumes for all samples and standards were set to 1 mL. Water was used as the weak wash solvent (700 mL) and a mixture of water and acetonitrile (1:1) was used as the strong wash solvent (200 mL).
Mobile phase components and gradient conditions are outlined in Table 1. Compounds were detected at a wavelength of 214 nm using a sampling rate of 20 points per second and a filtering constant of 0.1 seconds.
Calibration and Sample Preparation
A stock standard solution of acesulfame K (380 mg/ L), sodium benzoate (517 mg/ L), potassium sorbate (273 mg/ L), caffeine (253 mg/ L), and aspartame (500 mg/ L) was prepared in 30 percent acetonitrile. Six dilutions (in water) of the stock solution were prepared with concentrations outlined in Table 2. Calibration curves for all five compounds were generated from these analyses and fit to linear equations (see Fig. 2).
Samples were prepared by subjecting an aliquot (~ 20 mL) of the given soft drink to sonication for 5 minutes to remove dissolved carbon dioxide. The degassed sample was placed directly into 2 mL glass vials fitted with pre-slit septa. Small injection volumes (1 mL) minimized sample preparation by allowing the sample to be injected without a dilution step.
Results and Summary
Area counts from injections of a diet cola, lemonade and lemon-flavored diet cola (figures 3 to 5 respectively) were compared to the calibration curves (Fig. 2) and the amounts (mg/L) calculated for each compound (Table 3).
Retention times of all peaks were consistent with standard deviations of less than 0.10 seconds for all five compounds. The total cycle time for each analysis was 2 min 29 sec. Cycle time consists of the run time (1.25 minutes), system and column re-equilibration (45 seconds), injection time (29 seconds consisting of vial selection, sample draw, loop fill, and injection).
Using the same calculation as presented in the introduction, 10,000 samples can be analyzed in just 51 working days compared to the original 333 working days. This is 6.5 times faster, a substantial increase in sample throughput and subsequent laboratory productivity without sacrificing analysis quality.
Further, a significant reduction in the total amount of mobile phase used (and corresponding waste generated) can be realized; 160 liters total for the traditional method compared to 22.5 L for the UPLC method.
UPLC systems combined with UPLC BEH columns can provide a rapid analysis of soft drinks that is significantly faster than traditional HPLC techniques. The ability to reduce QC test costs while maintaining the quality of shipped manufactured product is desired in many food and beverage organizations. This enhanced capability and cost savings can be easily adapted to the analysis of a wide variety of compounds.