Performance Evaluation of an Integrated Analytical HPLC

As a routine analytical tool in the laboratory, there are many important performance requirements such as precision, reproducibility, and sensitivity for an analytical HPLC to achieve satisfactory use. Some of the performance characteristics of the Varian 920-LC (Varian, Inc., Melbourne, Australia) are discussed in this article.

Solvent delivery system

Figure 1 - Overlay of 10 repetitions of a 10% step gradient at 1 mL/min.

One of the critical performance criteria for an analytical HPLC is the ability to reliably and precisely deliver mobile phase to ensure reproducible separations of the compounds of interest. The solvent delivery system in the Varian 920-LC features a dual-piston pumping mechanism to deliver flows at pressures up to 5685 psi (392 bar) and flow rates from 0.001 mL/min up to 10 mL/min, and continuously deliver solvent virtually free of pulsations, eliminating the need for a pulse damper. Pulse dampers are used in solvent delivery systems to damp out residual pressure pulsations created by the pumping mechanism. Eliminating the pulse damper minimizes the system delay volume, allowing gradients to form precisely and without delay at the head of the column.

By using a low-pressure mixing design with four high-precision proportioning valves, the solvent delivery system is able to form a gradient from up to four different solvents. Figure 1 shows the reproducibility of gradient formation with an overlay of 10 separate runs of a 10% step gradient program at 1 mL/min, using a water/0.1% acetone mix added in 10% steps.

Precise, carryover-free sample injection

Figure 2 - Carryover of <0.01% caffeine injection measured at 254 nm.

The ability to make accurate, reproducible sample injections without carryover is the primary performance criterion for an autosampler in analytical HPLC. The Varian 920-LC autosampler features a needle-in-path design where the entire flow path is rinsed with mobile phase, minimizing carryover. An auxiliary needle wash port rinses the outside of the needle to further reduce carryover. In Figure 2, a 20-μL aliquot of 125 mg/L caffeine standard was injected and measured, followed immediately by a blank injection of the same volume. The retained caffeine in the blank was measured as carryover. As illustrated in Figure 2, the carryover is virtually undetectable, with results of <0.01% for caffeine shown. This is typical of results achieved using the autosampler.

The autosampler is able to accommodate up to 200 2-mL HPLC vials or three 96-well plates, providing high sample capacity for unattended automated sequence runs. A Peltier cooling accessory is available as an option when the instrument is to be used for the analysis of thermally unstable compounds.

Sensitive detection

For maximum detection limit performance, an HPLC must have sensitive and stable detectors. The Varian 920-LC offers a wide choice of absorbance detectors, including a dual-wavelength UV-VIS detector with a high light throughput and low noise electronics design that offers sensitive performance from 190 nm to 900 nm. A photodiode array (PDA) detector option is offered. The PDA detector has 1024 photodiodes with a deuterium lamp for operation in the UV and quartz iodide lamp in the visible wavelength range, guaranteeing the highest sensitivity and best resolution across the 190–950 nm range.

Figure 3 - A 10-pg injection of anthracene onto a Varian Pursuit™ 100 × 2 mm C8 column with a flow rate of 0.3 mL/min of H2O:ACN 15:85 gives a measured signal-to-noise ratio of 36.

In Figure 3, 10 pg of anthracene was injected onto a Varian 920-LC with a dual-wavelength UV-VIS detector according to the methodology of ASTM D 6156 97.1 Anthracene elutes at around 2.8 min, well away from the void volume, with an absorbance of approximately 270 μAU compared to a typical noise result of 7.5 μAU. The noise measurement was made according to the methodology of ASTM E 1657 98.2

For non-UV-absorbing compounds, a refractive index (RI) detector is also available for use. A sensitive fluorescence detector is offered as a second detector built into the Varian 920-LC.

Accurate and stable column temperature control

Figure 4 - Rapid warmup and stability and accurate column temperature of the Varian 920-LC column heater.

Table 1    -    Temperature accuracy and 2σ precision measured over 30 min on the column heater body and on the column itself

Accurately and precisely controlling the temperature of the column improves retention time reproducibility, which is vital for consistent long-term chromatographic performance (see Figure 4). The column heater in the Varian 920-LC uses a low delay volume preheater to efficiently transfer heat from the column heater to the mobile phase before it enters the column. As shown in Table 1, the preheater heats the mobile phase to within 1 °C of the set temperature before it enters the column, while the column heater controls the exterior temperature of the column.

Use of a preheater eliminates thermal gradients across the column, which can result in split peaks. This effect can occur with some column heater designs that only rely on convection or radiant heat. The warmup time from ambient temperature to 40 °C is less than 15 min and typically about 7 min.

Table 2    -    Column heater temperature accuracy

Figure 5 - Analysis of seven phenones in less than 1.5 min (a–g: acetophenone, propiophenone, benzophenone, valerophenone, hexanophenone, heptanophenone, octanophenone). Column: Varian Pursuit XRsUltra 2.8 C18; mobile phase: water/acetonitrile gradient, 45–100%; detection: UV at 245 nm; flow: 1.6 mL/min.

As shown in Table 2, along with the preheater, adequate thermal insulation ensures column temperature accuracy across the full temperature range of ambient + 5 °C up to 65 °C.

The combination of an accurate, low delay solvent delivery system; low-carryover sample injection autosampler; and use of a new column heater that provides precise column temperature control for reproducible retention time performance allows the Varian 920-LC to easily perform Fast LC on small particle size HPLC columns. Figure 5 shows the analysis of phenones using the HPLC system.

Conclusion

The Varian 920-LC is a robust, highly capable, integrated analytical HPLC. It features a dual-piston, low-pressure, quaternary gradient solvent delivery system with built-in four-channel Degasser™ to deliver virtually pulse-free solvent flows for accurate and highly reproducible gradient formation. It is supplied with a sensitive dual-wavelength UV-VIS or information-rich PDA detector. There is an option to add a fluorescence or universal RI detector built into the system. Provided as standard is an autosampler that is able to inject from up to 200 2-mL HPLC vials or three 96-well plates for complete unattended automated operation. The built-in, high-precision column with solvent preheater provides a thermally stable environment for the analytical column to deliver optimal retention time reproducibility, even in laboratories that do not have stable ambient temperatures.

The Varian 920-LC delivers reproducible results for a wide variety of analytical conditions from Fast LC using short 2-mm-i.d. columns packed with sub-3-μm particles through conventional 4.6-mm-i.d. columns packed with 3- and 5-μm particles.

References

  1. ASTM D 6156 97. Standard Practice for Use of High Performance Liquid Chromatographs.
  2. ASTM E 1657 98 (reapproved 2006). Standard Practice for Testing Variable Wavelength Photometric Detectors Used in Liquid Chromatography.

Mr. Russell is HPLC Product Manager, Ms. Truong is a Chemist, Mr. Buck is Development Engineer, and Mr. Button is European HPLC Product Manager, Varian Australia Pty. Ltd., 679 Springvale Rd., Mulgrave Victoria, Australia; tel.: +613 9566 1016; e-mail: glyn.russell@varianinc.com.

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