Compact capillary scale liquid chromatography (LC) instrumentation offers a platform for high performance separations with considerable reduction in solvent consumption, operating cost, and benchtop footprint when compared to analytical scale instrumentation. To enter more regulated environments, such as those often found in the pharmaceutical industry, systems must demonstrate their ability to operate under the conditions of existing pre-validated workflows. The United States Pharmacopeia (USP) provides monographs for the HPLC analysis of pharmaceutical compounds, as well as guidelines for adapting these monographs to analysis under modified HPLC conditions. Using USP 621 guidelines, a monograph for the analysis of hydrochlorothiazide was adapted for capillary scale analysis, passing system suitability while reducing solvent consumption by over 99%. For comparison, the analysis was also performed at analytical scale, with chromatographic figures of merit for both systems passing system suitability criteria. This work is an example that illustrates the ability of the Axcend Focus LC to satisfy USP monograph methods comparable to analytical scale chromatographic performance.
The United States Pharmacopeia (USP)–National Formulary (USP-NF) offers monographs for thousands of pharmaceutical compounds. These monographs are used around the world for sample validation. The conditions reported in these monographs (in this case, HPLC) can be modified for different column geometries, flow rates, and elution gradients if the results obtained are within validation specifications. To ensure the validity of a monograph post modification, USP has released guidelines in USP Chapter 621[1], outlining acceptable ranges for the parameters of the chromatographic process to be changed. The Axcend Focus LC is a compact capillary scale HPLC system that has been adopted for use in pharmaceutical laboratories. To demonstrate the validity of using this instrument for pharmaceutical analysis, a USP monograph for HPLC of hydrochlorothiazide[2] was adapted to capillary LC using USP 621 guidelines. Comparisons of system performance between analytical and capillary scales are given.
Column Dimensions
The particle size and or length of the column may be modified provided that the ratio of the column length (L) and the particle size (dp) remains within a range of -25% to +50% of the L/dp ratio given in the monograph. The resulting column adaptation parameters are listed below:
| Monograph column: | 4.6 x 50 mm column with 3.5 µm L1 particles | L/dp Ratio: 14.29 |
| Capillary column: | 0.3 x 50 mm column with 3.0 µm L1 particles | L/dp Ratio: 16.66 (+16.6%) |
| Analytical column: | 2.1 x 50 mm column with 2.6 µm L1 particles | L/dp Ratio: 19.23 (+34.6%) |
Flow Rate
When particle size and/or column diameter is changed, the flow rate must also be adjusted according to the equation:
Where F1 represents the flow rate listed in the monograph (mL/min), dc1 represents the internal diameter of the column listed in the monograph (mm), dp1 represents the particle size listed in the monograph (µm), F2 represents the adjusted flow rate (mL/min), dc2 represents the internal diameter of the column used (mm), and dp2 represents the particle size of the column used (µm).
| Monograph flow rate: | 1.0 mL/min |
| Capillary flow rate: | 0.0050 mL/min or 5.0 µL/min |
| Analytical flow rate: | 0.28 mL/min |
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