Achieve Faster Analyses on Any HPLC System Using Ultra II™ Columns

• Designed for scalability and optimal chromatography on any LC system.
• Comprehensive range of particle sizes—1.9µm, 2.2µm, 3µm, and 5µm.
• Available in traditional phases and unique chemistries for alternate selectivity.

Ultra High Pressure Liquid Chromatography (UHPLC) is arguably the most significant recent advancement in liquid chromatography. In the past few years, we have experienced an evolutionary leap in system performance that has altered our analysis time expectations for liquid separations. Implementing UHPLC technology can certainly produce faster separations and increase laboratory productivity, but adopting the technology requires substantial capital expenditures. Significant savings, in both time and solvent usage, are available to most labs right now, without the costly upgrade to UHPLC instrumentation and the associated hardware. These savings can be realized by evaluating and updating current methodologies that are time- and solvent-consuming. In this article, we will look at some simple steps and strategic column choices, which can significantly speed up analyses and decrease operating costs on existing systems.

Evaluate Selectivity for Speed and Savings

Although we often look exclusively to UHPLC for speed, by first utilizing column selectivity, many time-consuming methods can be updated to show immediate savings. For example, a common assay for famotidine and associated impurities can be improved by using new column chemistries. The traditional USP method calls for a high concentration, citrate buffered aqueous phase, an acetonitrile organic mobile phase, a traditional analytical scale C18 column (250mm x 4.6mm, 5µm), and a 45 minute gradient, resulting in a relatively slow analysis that requires a large solvent volume (Figure 1, Table I).

Figure 1: The conventional USP assay for famotidine and associated impurities is slow and expensive in terms of time and solvent use.

Peak List: Ret. Time (min.) 1. impurity A 2.833 2. impurity B 5.462 3. impurity C 7.905 4. famotidine 10.062 5. impurity D 11.874 Sample: Inj.: 10µL Conc.: 100µg/mL famotidine, 10µg/mL each impurity Sample diluent: methanol Column: conventional C18 Dimensions: 250mm x 4.6mm Particle size: 5µm Pore size: 60Å Conditions: Instrument: Shimadzu Prominence UFLCXR Mobile phase: A: 100mM sodium citrate trihydrate in water (pH 6.0):acetonitrile (93:7) B: acetonitrile Time (min.) %B 0 0 15 0 42 48 43 0 45 0 Flow: 1.5mL/min. Temp.: 35°C Det.: UV @ 268nm LC_PH0496

To speed up this method and reduce solvent consumption, we switched columns from a traditional C18 to a Restek Ultra II™ Aromax column (Figure 2). This novel bonded phase is both more retentive and more selective, which allows a smaller column length (150mm) and a faster gradient profile to be used. Since the selectivity of the Ultra II™ Aromax phase is enhanced with a methanolic mobile phase, the organic solvent was changed to methanol, which is currently a less expensive and more readily available solvent than acetonitrile. In addition, the new bonded phase allows the high concentration citrate buffer to be replaced with the lower concentration phosphate buffer. (Note: Citrate buffer in high concentrations has been shown to attack stainless steel in an HPLC and requires extensive, time-consuming flushing to remove.) By making a strategic column choice based only on selectivity, analysis time was reduced by nearly 70% and organic solvent use was reduced by nearly 60% (Table I).

Figure 2: Switch to a more retentive and selective Ultra II™ Aromax column and reduce analysis time by ~70% and solvent volume by ~60%.

Peak List: Ret. Time (min.) 1. impurity A 3.779 2. famotidine 8.264 3. impurity D 9.180 4. impurity C 9.911 5. impurity B 14.018 Sample: Inj.: 10µL Conc.: 100µg/mL famotidine, 10µg/mL each impurity Sample diluent: methanol Column: Ultra II™ Aromax Cat. #: 9607565 Dimensions: 150mm x 4.6mm Particle size: 5µm Pore size: 100Å Conditions: Instrument: Shimadzu Prominence UFLCXR Mobile phase: 20mM potassium phosphate (pH 2.5):methanol Time (min.) %B 0 5 15 35 Flow: 1.5mL/min. Temp.: 35°C Det.: UV @ 268nm LC_PH0495

Adjust Particle Size for Faster Analyses

Although choosing a column based on selectivity alone significantly improves analysis speed and reduces solvent costs, this method can be further optimized by scaling the analysis to a 3µm particle size (Figure 3). Ultra II™ Aromax columns are designed to be fully scalable and are available on a wide range of particle sizes to support labs interested in speeding up analysis times by switching to smaller diameter particles. While the pressure increase seen from this change was approximately two-fold, it is still well within the limits of what a normal HPLC will allow. The time and solvent savings due to scaling were both about 80% relative to the original method (Table I).

Figure 3: Switch to a 3µm Ultra II™ Aromax column and reduce both time and solvent usage—by over 80%.

Peak List: Ret. Time (min.) 1. impurity A 2.094 2. famotidine 4.460 3. impurity D 4.937 4. impurity C 5.334 5. impurity B 7.480 Sample: Inj.: 5µL Conc.: 100µg/mL famotidine, 10µg/mL each impurity Sample diluent: methanol Column: Ultra II™ Aromax Cat. #: 9607313 Dimensions: 100mm x 3.2mm Particle size: 3µm Pore size: 100Å Conditions: Instrument: Shimadzu Prominence UFLCXR Mobile phase: 20mM potassium phosphate (pH 2.5):methanol Time (min.) %B 0 5 8 35 Flow: 1.2mL/min. Temp.: 40°C Det.: UV @ 268nm LC_PH0494

Further savings can be realized by dropping the particle size to the pressure limit of the LC system and mobile phase. In this example we used a Shimadzu Prominence UFLCXR, a system capable of 660 bar maximum pressure, which allowed us to further scale the method to an intermediate 2.2µm particle diameter (Figure 4). With this system and column configuration, the analysis time is reduced to 5.5 minutes, including equilibration, and the mobile phase consumption is under 7mL per sample. This adds up to a time and solvent volume savings of over 90%—without any investment in specialized UHPLC equipment (Table I).

Figure 4: Scale down to a 2.2µm Ultra II™ Aromax column and cut analysis time and solvent use—by over 90%—without specialized UHPLC equipment.

Peak List: Ret. Time (min.) 1. impurity A 0.892 2. famotidine 2.082 3. impurity D 2.292 4. impurity C 2.517 5. impurity B 3.540 Sample: Inj.: 2µL Conc.: 100µg/mL famotidine, 10µg/mL each impurity Sample diluent: methanol Column: Ultra II™ Aromax Cat. #: 9607853 Dimensions: 50mm x 3.0mm Particle size: 2.2µm Pore size: 100Å Conditions: Instrument: Shimadzu Prominence UFLCXR Mobile phase: 20mM potassium phosphate (pH 2.5):methanol Time (min.) %B 0 5 4 35 Flow: 1.2mL/min. Temp.: 40°C Det.: UV @ 268nm LC_PH0493

Find the Analysis that Fits

So, how fast is fast enough? Admittedly, the example here could be scaled further by using an Ultra II™ Aromax in a 1.9µm UHPLC format. This is certainly an option, but the extra gains will be marginal and the cost of instrumentation may be prohibitive. In this example, we worked within the framework of our instrumentation and made a significant impact on the speed and cost of analysis. Each lab must carefully evaluate their own analytical needs and resources before determining how fast is fast enough.

Short-term savings of time and money can easily be achieved by optimizing outdated methods with novel column chemistries where appropriate. Evaluating selectivity is the first step in making strategic column choices; additional savings can be realized by choosing a particle size that best fits lab needs. New Ultra II™ LC columns are fully scalable, available in many phase chemistries, and designed to help labs adapt methods and speed up analysis times on any LC system.