Automated Column Selection and

Switching Systems for HPLC


by William Letter



The great popularity of high performance liquid chromatography (HPLC) systems today empowers chemists to determine the chemical purity of most types of samples. Advances in HPLC instrument design, computerized data analysis, and plotting software have added to the increased reliability and potential accuracy of modern instruments. However, the process of HPLC column selection and method development still consumes much time.

Often users must manually try different columns and mobile phase mixtures sequentially to find a satisfactory separation in a reasonable amount of time. Additionally, some samples require analysis using more than one column connected in series; they may even need a change in column type and mobile phase. When these activities are performed manually, chromatographers spend a great deal of time and effort. Several automated HPLC valving systems used for automated column selection and column switching can improve HPLC method development and sample analysis. Table I lists some of the obvious advantages of these systems.

TABLE I: Advantages of HPLC Column Selection and Switching Systems

Automated Column Selection   Automated Column Switching
  • No need to disconnect HPLC columns
  • No need to disconnect HPLC columns
  • More reproducible
  • More reproducible
  • Closed in-line system
  • Closed in-line system
  • Use of By-Pass line allows fast solvent changeovers and flow injection for LC-MS.
  • Backflushing and column bypass
  • Random Access to all HPLC Columns & By-Pass Line
  • Heart-cutting, 2D Multidimensional HPLC and fractionation possible
  • Use of second pump allows simultaneous analysis and column flushing with two columns
  • High throughput and shorter total analysis times


During HPLC method development, certain samples may require users to screen several column types to find the column that provides the best separation. This column selection technique often involves user intervention (e.g. stopping the pump, manually removing and re­installing a column or turning a valve, flushing, and equilibrating the system) before the next analysis can begin. Often these systems use valves to divert the solvent flow from one column to another column, as described elsewhere (1-16).

Modern electrically actuated column selection valves can be automated to perform unattended column selection, flushing, and re-equilibration. Actuator switching times of less than 100 ms allow column changeover to occur quickly and reliably.

An automated system can reduce the time it takes to develop new methods by screening one or more samples against a group of columns that contain different stationary phases. Mobile phase composition still can be varied as an important method development variable, but the stationary phase also can be varied with an automated system. When using automated systems, users are free to perform other work and monitor the instrument’s progress instead of returning to the system at the ideal time for installing and re-equilibrating new columns. The net result is a faster, more reliable screening process that easily allows tasks such as automated column testing, method development, performance verification, and chiral method development to occur in an after-hours environment, if desired.

The best automated column selection systems maintain each column hydraulically sealed in liquid, which greatly reduces the potential of chemical contamination and exposure to air. The LC SPIDERLING™ column selection systems hold up to nine HPLC columns and one by-pass line (4-6, 10-16). The system can select columns in any order and develop HPLC methods with little user intervention. They are available in heated/sub-ambient cooled as well as ultra-high pressure (600 Bar) versions. The by-pass line is included to facilitate solvent changeover and to permit flow injection of samples for liquid chromatography-mass spectrometry (LC-MS) analysis. Columns can be selected or skipped in any order and can be automatically flushed and re-equilibrated, if desired, at the end of each run. Automatically flushing columns after each use minimizes potential sample contamination and leaves the column in an appropriate mobile phase for use during the next analysis. I recommend using short lengths of narrow-bore connecting tubing (0.007" id.), fingertight column fittings and narrow bore valve ports (0.010" id.) to minimize band broadening in these systems. By automatically varying the column and mobile phase used, these systems allow automated HPLC method development of difficult to resolve compounds such as chiral racemates as well as automated analysis of known compounds in an automated fashion. This saves time and money and provides the chromatographer with time to work on other projects. These automated devices are believed to be the single largest improvement in modern HPLC method development to date.

Most column selection systems still require users to wait for columns to be flushed and re-equilibrated before they can begin a second analysis. However, if users want to repeat the same type of HPLC analysis repetitively on the same column type and have a second gradient pump available, then a new product allows im­mediate analysis after the first analysis is finished. The product is known as the Column Swapper (8 & 9) and was designed for repetitive HPLC sample analysis. It has a specially made, 10-port valve with very narrow ports to reduce dead volume and is controlled through simple HPLC control software which uses an ex­ternal timetable and contact closures to control the timing of the “column swap”. This version of Alternating Column Regeneration works well in labs which use the same method for several samples. This type of system can reduce the analysis time for repetitive HPLC analyses by 50%. The Column Swapper™ provides all of this ingenious functionality in one easy to use package.



If a sample requires purification, concentra­tion, and analysis using two or more stationary phase types, chromatographers frequently use multidimensional chromatography sometimes called LC-LC or ‘2D’ HPLC. Multidimensional column chromatography is a powerful technique for the separa­tion and cleanup of multi-component mixtures. In this technique, fractions from one chromatographic column are transferred selectively to one or more secondary columns for additional separation. This technique has been popular in preparative HPLC for many years, but has been done off-line by most analytical chromatographers. 

Multidimensional chromatography can be used for:

·         trace enriching of selected analytes,

·         improving the resolution of complex sample portions (maximum resolution can be achieved by using different modes, stationary phases, and mobile phases)

·         increasing sample throughput by the use of heart-cutting, backflushing, front- or end-cutting, and recycle chromatography methods.

In HPLC, multidimensional chromatography can be performed off-line or in-line. For the off-line technique, users collect fractions of solute at the detector exit from the first col­umn (often in a glass vial) and re-inject the collected fraction onto the secondary column. This approach usually works fine, unless the job is a repetitive task. If the task must be repeated, an in-line system is much more effective. For the in-line approach, better known as column switching, chromatographers must couple two columns using high-pressure switching valves that either trap defined volumes or the collected samples, usually in a loop, and direct them to the second column (a process called heart-cutting). Alternatively, switching valves can be plumbed to divert the mobile phase containing the desired solutes from the first column to the second column for defined periods of time (a process sometimes called on-column concentration).

Others have described numerous innovations in the development of column-switching systems (17-28). Chromatographers can use column-switching to select specific columns for analysis or connect more than one column in series. The systems also can backflush specific sample components from one column to waste, which leaves only the peak of interest on the second column. During peak transfer, sample volume can be decreased between columns through such tricks as using a column with a larger volume than the first as the second column. In all cases, when developing 2D methods, developing a solid HPLC method in the first place which provides for excellent, narrow peak shapes to minimize sample volume during fractionation or cutting will usually provide the best results.

Our fully automated 2D HPLC Column Switching module consists of several high quality, pre-plumbed valve modules for column switching (26). This system can be used for conventional HPLC analysis, and it can perform more advanced functions such as backflushing, heart-cutting, trace enrichment (on-column concentration), and front- and end-cutting when needed. The in-line design of this type of system also can reduce sample contamination and loss. The column-switching system can be fully automated using the built-in contact closures with your software’s timed-event tables.


Sample Peak Recycle Chromatography, “Column Leap Frogging”

When a column’s stationary phase only partially resolves a sample or pair of peaks (i.e. chiral HPLC method development) a technique know as recycle chromatography can be used to increase the amount of stationary phase available to the sample. Popular in preparative HPLC fields where the sample is often recycled directly back through the pump head; recycle chromatography in analytical HPLC by this method would be impractical due to sample dilution. A column switching solution which works well for analytical scale columns, the LC Sample Peak Recycler™, was developed in the 1990’s and utilizes the novel idea of “column leap frogging” (27-29). To avoid sending the sample through the analytical pump head the LC Sample Peak Recycler™ improves the resolution of partially resolved peaks by repeatedly recycling the sample through two identical columns. The novel valve switching product passes the sample peaks from one column to the next in a leap frog manner by directing the sample onto the next column at just the right moment (as monitored in real time using your detector) using a contact closure which can be easily controlled by most HPLC software controllers. The sample can be passed from column to column over and over simulating a much longer column without the added cost or prohibitive backpressure. The device can be of great value with difficult to resolve chiral samples.



With the increasing popularity of LC-MS and LC-MS-MS systems, laboratory workers are paying much more attention to sample throughput and source cleanliness. An improvement to an existing two-position column-switching valve that is equipped with a microprocessor (30) has been developed specifically for LC-MS users. The device uses the output from the HPLC’s  UV detector to monitor the absorbance signal and divert the solvent flow away from the MS source when the UV signal exceeds a specific, user-defined value (between 1 and 2000 mAU). Diverting the flow to waste, instead of sending the highly concentrated sample to the MS source, reduces the potential contamination of the mass spectrometer. As an option, the device allows a second pump to supply the MS source with solvent while the main flow is diverted away to waste. For GLP documentation purposes, the unit automatically generates a chart mark output each time the microprocessor switches the valve diverter. This system is usually referred to as the “LC-MS Watchdog” as it provides a great way for lab managers to protect an Open-Access LC-MS system from expensive source contamination.



Chromatographers can use column-selection and column-switching valves in various ways to improve the development and performance of HPLC methods. Multiport, high-pressure valves coupled to electric valve actuators allow many different tools to be constructed for solving diverse applications. These devices can save valuable time by automating tasks that are performed manually.

The use of column selection systems to screen, test, flush, equilibrate, and develop methods automatically can save time and provide more data for decision making. When necessary, using in-line column switching systems also can reduce the amount of wasted sample and improve the reliability of the methods used. As a bonus, these column-selection and -switching tasks often can be performed after hours, which saves even more time in the laboratory and increases overall productivity.



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©Copyright January 2008, Chiralizer Services, LLC. All Rights Reserved.

NOTE: Portions of this article were originally published in LC-GC, Volume 15, Number 6, pp 508-512, June 1997, but is now presented here in a newly revised and web updated form by the author in 2008. You may copy or link to this document or web page as long as you include this original web link address AND reference our company as the owner/author of this copyrighted material.