Optimize Selectivity & Efficiency in UHPLC Separations

With More Stationary Phase Choices on 1.9µm Pinnacle™ DB HPLC Columns

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• Largest variety of stationary phases for UHPLC.
• Faster analyses, uncompromised chromatography.
• 100% Restek manufactured—from base silica to final packed column.

Since the late 1960s, when modern high performance liquid chromatography (HPLC) became a viable tool for practicing chemists, continual advancements have been made in column technology. Over time, the trend has been to pack columns with smaller particle sizes, which necessitates increasing the pressure capabilities of our instrumentation. These trends have brought us to where we are today—Ultra-High Performance Liquid Chromatography (UHPLC). UHPLC is a milestone in the evolution of LC in that columns packed with <2 μm particles, used in conjunction with instrumentation capable of handling the resulting high back pressures, make extremely fast and efficient separations possible. UHPLC is a very powerful tool for today’s chromatographer, as it can significantly increase the efficiency of a chromatographic separation. In addition, the wider range of usable flow rates makes high-speed separations possible. However, in light of this new technology, it is important that we do not forget the importance of selectivity. In this article, we will look at the principles of resolution and how we can use these concepts, namely selectivity, to maximize the benefits of UHPLC.

In past articles we have discussed the physical advantages that are driving interest in small particles, mainly the influence of particle size on usable flow rates and peak efficiency. Although small particles have made faster separations possible, the ultimate goal behind chromatography is still analyte resolution and particle size is only one contributor to this goal. Resolution is the result of three cumulative terms: selectivity, efficiency, and retention. How well we resolve our analytes, and how quickly we do it, depends upon our ability to control these 3 factors. As we can see through the resolution equation (Equation 1), mathematically, the selectivity term has the greatest effect on resolution, indicating that resolution is largely a function of selectivity. Selectivity, in turn, is governed predominantly by analyte interactions with both the stationary and mobile phases. UHPLC, through the use small particle columns, does maximize efficiency (i.e. theoretical plates), but the stationary phase is still the most important consideration when attempting to resolve mixtures of compounds. Ideally, a stationary phase that produces optimum selectivity or allows for the resolution of compounds in a timely manner should be selected.

Equation 1:  The resolution equation indicates selectivity has the greatest influence on resolution.

Previously, some advantages of selectivity in specific separations have been noted. For example, the use of the unique Biphenyl stationary phase has shown excellent selectivity for aromatic or fused ring compounds. When using the Biphenyl stationary phase and combining it with the heightened efficiencies of the Pinnacle^®: DB 1.9 μm particle size column, we can produce highly selective and fast separations of steroids (Figure 1). A 1.9 μm Pinnacle^® DB Biphenyl column can separate a test mix of 7 hormones in under 2 minutes, a feat not possible through C18 selectivity.

Another example of unique selectivity available on a 1.9 μm particle size column is the PFP Propyl (pentafluorphenyl propyl) stationary phase for halogenated drug compounds. This phase is very selective and retentive for organohalogens or other compounds containing basic or electronegative functionalities. To demonstrate heightened selectivity for halogenated drug compounds, we assayed a test mix of 8 benzodiazepines and 2 metabolites, a mix commonly assayed on a C18 colum, in just over 4 minutes with complete resolution (Figure 2). To get the same level of selectivity from a C18 column, a shallower gradient would be needed, prolonging the analysis time. Since the selectivity of the 1.9 μm Pinnacle^® DB PFP Propyl column elutes the benzodiazepines in quick succession, a simple gradient still allows for the earlier elution of the more polar metabolites, while maintaining a fast overall run time.

Restek is committed to giving the practicing chromatographer choices, and has therefore sought to deliver the widest selection of stationary phases available with <2 μm particle sizes. The goal of chromatography is always to resolve compounds of interest in the fastest time possible. By combining the benefits of UHPLC with Restek’s line of unique stationary phase choices, faster separations become a reality.

Figure 1:   Restek’s 1.9 μm Pinnacle® DB Biphenyl columns are highly selective for steroids, making an extremely fast and selective analysis.

Peaks 1. Estriol 2. 17β-Estradiol 3. 17α-Estradiol 4. Ethynyl estradiol 5. Testosterone 6. Estrone 7. Norethindrone LC_PH0453 Column Pinnacle® DB Biphenyl (cat.# 9409252) Dimensions: 50 mm x 2.1 mm ID Particle Size: 1.9 µm Pore Size: 140 Å Temp.: 30 °C Sample Diluent: Water:methanol (50:50) Conc.: 100 µg/mL Inj. Vol.: 1 µL Mobile Phase A: Water B: Methanol Time (min.) %B 0 30 1 30 3 70 Flow: 0.8 mL/min. Detector UV/Vis @ 220 nm

Figure 2:   Fast, selective analysis of benzodiazepines is made possible by combining the speed of UHPLC with the enhanced selectivity of the 1.9 μm Pinnacle® DB PFP Propyl column.
1.9 μm Pinnacle® DB PFP Propyl
Peaks Conc. (µg/mL) 1. 7-amino Clonazepam* 20 2. 7-amino Flunitrazepam* 20 3. Bromazepam 100 4. Oxazepam 100 5. Lorazepam 100 6. Clonazepam 100 7. Nitrazepam 100 8. Nordiazepam 100 9. Flunitrazepam 100 10. Diazepam 100 * metabolite LC_PH0452 Column Pinnacle® DB PFP Propyl (cat.# 9419212) Dimensions: 100 mm x 2.1 mm ID Particle Size: 1.9 µm Pore Size: 140 Å Temp.: 40 °C Sample Diluent: starting mobile phase (80:20 A:B) Conc.: as listed Inj. Vol.: 1 µL Mobile Phase A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile Time (min.) %B 0 20 1 20 6 80 Flow: 0.6 mL/min. Detector UV/Vis @ 254 nm
1.9 μm Conventional C18
LC_PH0451 Column Conventional C18 Dimensions: 100 mm x 2.1 mm ID Particle Size: 1.9 µm Pore Size: 140 Å Temp.: 40 °C Sample Diluent: Starting mobile phase (80:20 A:B) Conc.: µg/mL As listed Inj. Vol.: 1 µL Mobile Phase A: 0.1% Formic acid in water B: 0.1% Formic acid in acetonitrile Time (min.) %B 0 20 1 20 6 80 Flow: 0.6 mL/min. Detector UV/Vis @ 254 nm

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