In this application we demonstrate the analysis of coffee and coffee products according to DIN methods. The performance of the analysis is shown for linearity, limit of detection (LOD), limit of quantification (LOQ), retention time, and area precision. The performance is also shown for solvent-saver columns with 2.7 µm superficially porous particles.
The measurement of some compounds inherent in coffee products by means of high perfomance liquid chromatography (HPLC) with UV absorbance or fluorescence detection is standardized in the DIN ISO methods:
1) Agilent ZORBAX Eclipse Plus C18, 4.6 × 150 mm, 5 µm. Flow rate 1.0 mL/min.
2) Agilent Poroshell 120 EC-C18, 3.0 × 150 mm, 2.7 µm. Flow rate 0.43 mL/min.
3) Agilent Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 µm. Flow rate 0.43 mL/min.
For all compounds calibration curves were created by means of a UV absorbance detection with the exception of ochratoxin, where a fluorescence detection (FLD) was used. The calibration levels were measured under standard HPLC conditions (column 1) as described in the DIN literature. The dilution series was conducted until the limit of quantification (LOQ) was reached at a signal-to-noise (S/N) ratio of 10 and the limit of detection was calculated for a S/N ratio of 3 (Table 1). Typically, the LOQs are in the lower µg/L range, for ochratoxin in the lower ng/L range. From multiple injections, the relative standard deviation (RSD) values of the retention time, below 0.07%, and the area RSD, below 0.2%, were calculated.
Table 1: Performance data for different analytes and analysis methods.
As an example, the separation of isomeric chlorogenic acids from a roasted coffee sample under standard HPLC conditions is shown in Figure 1a. The isomers are separated over a run time of 30 min. The experiment was reaped after transferring the method to column 2 with a flow rate of 0.43 mL/min. For the calibration, similar linearity was found but the LOQ and LOD were lower on the 2.7 µm column. This was due to the better separation performance, showing narrower and sharper peaks with improved S/N ratio enabled by the 2.7 µm superficially porous particles (Table 1, row B; Figure 1b). In addition, this column saves 57% of solvent. To improve the efficiency, column 3 was used. This decreases the total run time by a factor of three and allows increasing sample throughput by a factor of three (Figure 1c).
Figure 1: Analysis of isomeric chlorogenic acids on different column dimensions.
The possible improvement of standard DIN methods for the analysis of coffee and coffee products is shown. The throughput could be increased by a factor of three while saving 57% of solvent.
(1) E. Naegele, "Determination of Caffeine in Coffee Products According to DIN 20481" Agilent Technologies Application Note, publication number 5991-2851EN (2013).
(2) E. Naegele, "Determination of Chlorogenic Acid in Coffee Products According to DIN 10767" Agilent Technologies Application Note, publication number 5991-2852EN (2013).
(3) E. Naegele, "Determination of Methylcafestol in Roasted Coffee Products According to DIN 10779" Agilent Technologies Application Note, publication number 5991-2853EN (2013).
(4) E. Naegele, "Determination of Ochratoxin A in Roasted Coffee According to DIN EN 14132" Agilent Technologies Application Note, publication number 5991-2854EN (2014).
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