Environmental Applications of a UHPLC System: The Evolution of Chromatography

Ultrahigh-performance liquid chromatography (UHPLC) has brought two important innovations to LC-MS  analysis. First is the use of 1.8-µm columns, which provides an increase in plate number from the 5-µm columns of about a factor of two.This is important for environmental applications where complex matrices are encountered, such as pharmaceuticals in wastewater analysis. However, the use of 1.8-µm columns requires higher-pressure pumps operating at pressures that exceed 600 bar on a routine basis, which is the range commonly used for UHPLC. Secondly, the 1.8-µm column allows the use of Rapid Resolution Liquid Chromatography (RRLC) (Agilent Technologies, Santa Clara,CA) and even ultrafast chromatography, where runs may be less than 1 min in length. The high flow rates required for these analyses create pressures on the order of 800–1000 bar, or more, and are clearly in the range of UHPLC.

The demands of high sample throughput in short time frames have given rise to high efficiency and fast liquid chromatography using the 1.8-µm reversed-phase columns. Fast chromatography has become a necessity in laboratories that analyze hundreds of samples per day or those needing short turnaround times. Using RRLC, results of a sample batch can be reported in a few hours rather than a few days. In both the water quality and food industries, regulatory laboratories produce validated results in less than an hour so that water treatment may proceed or vegetable shipments can be released the same day they are measured or produced. The end result is greater productivity for users and greater cost efficiency for the reporting laboratory. Thus, productivity is improved by shortened analysis time, which typically requires UHPLC. The definition of Rapid Resolution Liquid Chromatography is simple. Liquid chromatographic separations that are less than 10 min are fast, and separations less than 1 min are widely known as ultrafast.1

The other aspect of UHPLC is the increased peak capacity available when longer columns with 1.8-µm packing are used. It is now possible to have almost 300 times greater peak capacity, which is a valuable asset to unknown analysis in wastewater and other environmental applications such as pesticide screening. Finally, the UHPLC system should be robust and capable of both high pressure and high flow (>1 mL/min at pressures up to 1200 bar) to carry out both Rapid Resolution and normal flow chromatography with high peak capacity. Specially designed for pressures to 1200 bar (18,000 psi), 1.8-µm columns from Agilent Technologies give a variety of phases (C8, C18 in both Stable Bond and ZORBAX Eclipse Plus formats). These are useful for difficult water samples, as this article will show, including improved peak capacity for U.S. EPA Method 1694 for pharmaceuticals in wastewater. They are also useful for rapid resolution of pharmaceuticals and pesticides using both triple quadrupole mass spectrometry as well as liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS).

Experimental

The work shown here was carried out by Drs. Imma Ferrer and Michael Thurman at the Center for Environmental Mass Spectrometry at the University of Colorado, Boulder, using the Agilent 1290 Infinity LC system and both the Agilent 6430 triple quadrupole LC-MS and Agilent 6220 accurate-mass time-of-flight LC-MS system.

Columns

Table 1    -    Columns used in this study

Two different columns were tested for Rapid Resolution High-Throughput (RRHT) analyses, including the high-pressure (1000 bar) 1.8-µm particle sizes. Table 1 lists the columns tested in this work and their theoretical plates.

Chromatographic and mass spectral conditions

The Agilent 1290 Infinity LC was used for all UHPLC chromatographic separations, and the Agilent 1200 Series SL was used for the standard U.S. EPA Method 1694. The conditions were as follows for each figure.

Figure 1 - Reduction of analysis time from 30 min to 10 min using the Agilent 1290 Infinity with UHPLC for Group 1 pharmaceuticals in U.S. EPA Method 1694.

In Figure 1a, the liquid chromatograph was the Agilent 1200 Series SL. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 30 min with a 5-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 4.6 mm × 150 mm, 3.5 µm. Peak widths at the base were 15–18 sec with peak capacity of 100. Maximum pressure was 75 bar.

The mass spectrometer was the Agilent 6410 triple quadrupole LC-MS system in electrospray positive mode with three time segments in multiple reaction monitoring (MRM) mode. There were two transitions per compound with 10-msec dwell time for each transition. The compounds were Group 1 of U.S. EPA Method 1694. (See Ref. 2 for further detail on compounds and their transitions.)

In Figure 1b, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 10 min with a 1-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 50 mm, 1.8 µm. Peak widths at the base were 5–6 sec with peak capacity of 100. Maximum pressure was 375 bar.

Figure 2 - Increased peak capacity showing the separation of the entire list of U.S. EPA Method 1694 pharmaceuticalsplus 15 new compounds for a total of 90 pharmaceuticals in less than 20 min using a ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8-µm packing material with UHPLC using the Agilent 1290 Infinity LC. Peaks were 5–6 sec wide and peak capacity was 200.

The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode. There were two transitions per compound with 10-msec dwell time for each transition. The compounds were Group 1 of U.S. EPA Method 1694. (See Ref. 2 for further detail on compounds and their transitions.)

In Figure 2, the liquid chromatograph was the Agilent 1290 Infinity LC. The gradient was from 10% acetonitrile/water to 100% acetonitrile in 20 min with a 2-min hold time. The flow rate was 0.6 mL/min. The column was the ZORBAX Eclipse Plus-C18, 2.1 mm × 100 mm, 1.8 µm. Peak widths at the base were 5–6 sec with peak capacity of 200. Maximum pressure was 750 bar.

The mass spectrometer was the Agilent 6430 triple quadrupole LC-MS in electrospray positive mode with one time segment in MRM mode. There was one transition per compound with 10-msec dwell time for each transition. The compounds were Groups 1–4 of U.S. EPA Method 1694, plus 15 additional pharmaceuticals. (See Ref. 2 for further detail on compounds and their transitions.)

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