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Go Ballistic...with HALO-5 Fused-Core 5-mm HPLC Columns!


Why Use Ballistic Gradients?

Many practitioners of LC-MS are very familiar with the term “ballistic gradients”, and use them quite often in their work.  The term “ballistic gradient” typically refers to a fast or ultra-fast gradient (often with a gradient time of 0.5 to 5 minutes), which uses a large range of organic modifier (often 5-95% or 0-100%), a narrow 2.1 mm ID column, and a relatively high flow rate for that diameter (³ 1.0 mL/min).  Such gradients are used for assessing identity and purity of compound libraries, new product candidates, reaction mixtures, and for any type of situation for which high throughput is required or desired.  They are also useful and practical for situations in which a lower resolution or an “at-line” gradient method is needed for product or reaction progress characterization.  Moreover, with proper care, one can translate a longer method on a larger ID and longer column to a high-throughput method for LC-UV and/or LC-MS.  See Reference 1 for more information.

 

How to Get Maximum Performance with Ballistic Gradients

Usually, those applying fast gradients with LC-MS or even LC-UV already have instrumentation designed or plumbed for such purposes.  However, that’s not always the case, and it’s worthwhile to review some of the things you should consider when you’re trying to obtain maximum performance from high throughput separations.

 

Delay Volume

For ultra-fast gradients (0.5–2 minutes) using flows rates of 1 mL/min or higher, choose an instrument with a delay volume of 0.5 mL or less to minimize the time it takes for the gradient to reach the column.  With some instruments and autosamplers you can use an injector program to delay the injection a predetermined amount of time after the gradient program starts to reduce the “effective delay volume”.  One can calculate the desired injection delay time from the measured gradient delay volume, and the flow rate.  However, for some instruments, the delay volume is a function of column backpressure, which is dependent on column length, particle size, flow rate, and temperature (viscosity).

Figure 2 

Extracolumn Dispersion

The usual parameters that affect extracolumn dispersion must also be considered when optimizing performance, including: 

  • tubing ID and length (0.005” ID and minimal length)
  • injection volume
  • sample solvent strength and viscosity (relative to starting mobile phase)
  • flow cell volume (for LC-UV runs, and for LC-UV detection preceding LC-MS)
  • detector time constant and data rate (LC-UV)
  • LC-MS scan rate - Extracolumn volume does not equate directly to extracolumn dispersion, and, depending on the instrument and design, but extracolumn dispersion (also known as band spreading and peak variance) can increase with flow rate (Figure 1 on the left from Reference 3).  In addition, extracolumn dispersion is dependent on the analyte(s), organic modifier choice, organic modifier composition and column temperature for both isocratic and gradient analyses.

 

Figure 1  Extra-column variance (µL2) vs. flow rate on several HPLC and UHPLC Systems.  It is beyond the scope of this LabNote to describe how these results were obtained by the authors.  Please see Reference 3 for a detailed description.  For the upper plot, results were obtained for naphtho[2,3-a]pyrene using 100% acetonitrile; for the lower plot results were obtained using 4-tertbutylphenol using a mobile phase of 65:35 (v/v) methanol/water.

The observed peak variance in µL2 can also differ significantly among various instrument models and configurations of HPLC and UHPLC instrumentation. As an example, some results published by Guiochon and Gritti in Ref. 3 are shown in Figure 1. Note that the plots of dispersion vs. flow rate are linear-log plots. Other instruments have been commercialized since the publication of Ref. 3, such as the Acquity H and I Class instruments, which have very low extracolumn volume and dispersion.

Figure 2

Advantages of HALO-5 for Ballistic Gradient Analyses

Our sales results breakdowns indicate and customer feedback has told us that the HALO-5 columns are being used more frequently for high throughput applications.  In particular, the shorter lengths (20, 30, and 50 mm) in 2.1 mm diameter are becoming more and more popular among LC-MS users.  They have found that the HALO-5 columns are very rugged for such applications, and allow them to use their HPLC systems and mass spectrometers that cannot handle the very narrow peaks produced by sub-2-mm columns.  HALO-5 particles have been shown to deliver reduced plate heights (h) of 1.2 at their optimum flow rate and linear velocity, and they also have a relatively flat van Deemter plot (see Figure 2, resolution vs. flow rate).  Moreover, HALO-5 columns have the much lower back pressure of a 5-mm column with the performance of a 2.5–3.0 mm particle.


For HPLC users, HALO-5 columns, with their 2-mm frits, provide excellent performance and superior ruggedness compared to UHPLC and UPLC columns for high-throughput applications and for bioanalyses by LC-UV and LC-MS.  In laboratory comparisons between a 2.1 x 50 mm sub-2-mm column and the same size HALO-5 column, a flow rate of 2.0 mL/min at 40°C (acetonitrile/water) could be used with the 2.1 x 50 mm HALO-5 column (Figure 3), with a worst-case backpressure of 210 bar (3050 psi).  With the sub-2-mm column, a flow rate of only 0.75 mL/min produced a worst-case backpressure over 420 bar (which exceeds the backpressure limit for HPLC systems) under the same conditions.  

Figure 3

In addition, the lower backpressure of the HALO-5 column allows you to use a longer column in place of the shorter column to obtain additional resolution for difficult samples. This advantage is shown below (Figure 4) in which a 2.1 x 150 mm HALO-5 column is used at 1.0 mL/min (280 bar/4070 psi) with a longer gradient time to pull apart additional peaks in the same sample shown previously in Figure 3. 

Figure 4

HALO-5 columns are currently available in the following phases:  

  • C18
  • C8
  • Phenyl-Hexyl
  • PFP
  • ES-CN
  • HILIC
  • Penta-HILIC

The latter two phases are quite useful for LC-MS applications for analyses of basic analytes and very polar analytes.  HALO-5 RP-Amide and Peptide ES-C18 phases are expected to be available later in 2013.

For more information on HALO-5 HPLC and HALO 2.7 UHPLC columns, please visit our web site. 

References

  1. “Bridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused-Core® Columns”, Poster, PittCon 2012, Stephanie. A. Schuster and Thomas. J. Waeghe. 
  2. European Journal of Pharmaceutics and Biopharmaceutics 68 (2008) 430–440, “Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particles. Part II: Gradient experiments.  Davy Guillarme, Dao T.T. Nguyen, Serge Rudaz, and Jean-Luc Veuthey.
  3. Journal of Chromatography A, Volume 1217, Issue 18, 30 April 2010, Pages 3000–3012.  “Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments”, Fabrice Gritti, Carl A. Sanchez, Tivadar Farkas, and Georges Guiochon.
  4. Journal of Chromatography A, 1216 (2009) 5979–5988, Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-µm porous particles; Kenneth J. Fountain, Uwe D. Neue, Eric S. Grumbach, and Diane M. Diehl, Waters Corporation.

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