LCGC North America
We recently introduced a new series of UHPLC columns with a particle size of 1.6 µm. Here we explain how to transfer an existing HPLC method to a new UHPLC method using polyphenols as an example.
Figure 1: Chromatogram obtained as the first step. Standards: 1. Puerarin 2. Baicalin (37.93)* 3. Resveratrol (2.00) 4. Daidzein (2.75) 5. Quercetin (3.31) 6. Biochanin A (26.69) 7. Curcumin (4.85) 8. Ipriflavone (16.70) *( )s indicate separation factor.
We previously published an HPLC method on Application Data No. 112 (https://develosil.us/wp-content/uploads/DN112-0519-Analysis-of-Polyphenols-HPLC.pdf). Entering parameters of the method and the specification of the new UHPLC column in a widely available method transfer software program generated an initial gradient table (Table I). The software suggested a flow rate of 0.375 mL/min; this was adjusted to 0.5 mL/min, the optimum flow rate for this column.
The first thing to decide is the type of detection. If using mass spectrometry, only volatile mobile phase modifiers such as formic acid can be used. In the case of UV detection, acetic acid, formic acid, phosphate buffers, and others can be used. We chose 0.1% formic acid, so that either detection method may be used. Since it can be prepared with a pipette alone, it has the advantages of time and less human error.
The tailing factor and the separation factor obtained using different acids in the mobile phase are shown in Table II for each analyte. Quercetin is known to have a tailing tendency with formic acid, and may also show carryover due to strong ligating properties. Although 0.1% formic acid shows slight tailing with a tailing factor of 1.43, two other mobile phases showed even better results. Considering LC–MS use, we chose 0.1% formic acid as the first candidate. For better peak shapes, 0.08% formic acid + 0.02% TFA is an option.
Considering that baicalin and resveratrol have very closely eluted peaks, 0.1% formic acid showed the best separation factor. After adjustments to allow for elution of ipriflavone, we set the final gradient conditions as shown in Table III.
Mobile phase: A) 0.1% formic acid in water, B) 0.1% formic acid in acetonitrile. Conditions: Column: Develosil UHPLC C18, 1.6 µm Size: 2.0 × 50 mm; Temperature: 40 °C; Detection: UV at 260 nm; System: UHPLC with a mixer of 100 µL
Mobile phase: A) 0.1% formic acid in water, B) 0.1% formic acid in acetonitrile
Develosil USA
10060 Carroll Canyon Rd, Suite 100, San Diego CA 92131
Tel and Fax: (858) 800-2433
Website: www.develosil.us
Synthetic Peptides: Chromatographic Methods for Separation, Quantification, and Characterization
May 9th 2024Peptides are versatile molecules that have a wide range of applications in biotechnology, medicine, and research. They can be used as drugs, diagnostics, biomarkers, or tools to study cellular processes. However, peptides also pose unique challenges in terms of analysis and purification due to their complexity, diversity, and sensitivity.
Inside the Laboratory –– Women in Separation Science Edition
April 25th 2024Inside the Laboratory is a joint series with LCGC and Spectroscopy, profiling analytical scientists and their research groups at universities all over the world. This series spotlights the current chromatographic and spectroscopic research their groups are conducting, and the importance of their research in analytical chemistry and specific industries. In this “Inside the Laboratory –– Women in Separation Science” special edition PDF, we profile four industry leaders and the impact that their research is having in specific fields such as environmental analysis, forensics, national security, and drug development.