The Column - August 2008 - (Page 23)

August 2008 www.thecolumn.eu.com The Column Kittell, Van Soest and Arnold Transferring Existing HPLC Methods to MicroLC Justin Kittell, Remco Van Soest and Don Arnold, Eksigent, Dublin, California, USA. Transferring existing HPLC methods to MicroLC allows for faster separations due to lower backpressures. In this article, a comparison of conventional and MicroLC analyses is described. Introduction MicroLC uses µL/min flow-rates on 0.3 to 0.5 mm i.d. columns. MicroLC offers a number of advantages to chromatographers over conventional HPLC approaches. An important advantage of using small i.d. columns is that these columns provide equal performance to conventional 4.6 and 2.1 mm i.d. columns but with reduced backpressure requirements. Also, by reducing solvent usage by 100 L/year* per instrument, thousands of dollars a year can be saved in solvent, storage and waste costs as well as making a significant contribution to green chemistry initiatives. Eksigent has developed a tightly integrated MicroLC system that uses continuous feedback to deliver low flow-rate gradients accurately and precisely. The system design removes the need for pressure dampeners and large volume mixers, thereby reducing delay volumes and re-equilibration times. As an additional benefit of microflow operation, the pump design significantly increases pump lifetime, making the fluid delivery system nearly maintenance-free and increases overall system reliability to levels expected from state-of-the-art HPLC tools. The smaller i.d. columns used in MicroLC are packed with the same stationary phases that are common in conventional 2.0 to 4.6 mm i.d. columns. Separation methods developed on conventional columns are adapted for use with smaller i.d. columns by scaling down the flow-rates and injection volumes according to the cross-sectional area of the column. Using columns packed with the same 3 µm stationary phase, the flow *Based on 1 mL/min, 8 hours/day and 200 days/year. Authors: Justin Kittell, Remco Van Soest and Don Arnold E-mail: jkittell@eksigent.com resistance measured using the same mobile phase face velocity in a 0.3 150 mm i.d. column is less than half that of a 4.6 150 mm i.d. column. The methods are seamlessly transferred as demonstrated by the similar retention times, resolution and performance from the smaller i.d. columns but with significantly less backpressure applied. Experimental Methods Method transfer: For the evaluation of the scale-down of a gradient separation method from a 4.6 to 0.3 mm i.d. column, a test sample composed of six compounds (caffeine, indomethacin, methyl paraben, ketoprofen, butyl paraben and amitriptyline) with a final concentration of approximately 0.1 mg/mL each was used. The gradient separation conditions used on both systems were: Column stationary phase: ProntoSil C18CL, 3 µ, 120 Å Column length: 150 mm Column temperature: Ambient Mobile phase A: 0.1% TFA Mobile phase B: acetonitrile w/0.1% TFA Gradient: 2–95% B, linear, 4 minutes Detection wavelength: 236 nm To adapt the gradient methods developed with a conventional 4.6 mm i.d. column for a smaller i.d. column, only the flow-rates and injection volumes were scaled down according to the ratio of the cross-sectional areas of the two columns. The ExpressLC-100 (Eksigent Technologies), designed 23 Stockbyte/Getty Images http://www.thecolumn.eu.com

Table of Contents for the Digital Edition of The Column - August 2008

The Column - August 2008
Contents
Pesticide Residues in Fruit and Vegetables - From Avocados to Zucchinis
News
Incognito
Market Trends and Analysis
Tips and Tricks: GPC/SEC What are The Applications for Polymer Reference Standards?
Transferring Existing HPLC Methods to MicroLC
Q&A

The Column - August 2008