Ion exchange chromatography (IEX) complements the more common size exclusion chromatography (SEC) by offering a different approach to sample separation. While SEC focuses on separating molecules based on their hydrodynamic volume, IEX uses ionic charge as its separation principle. This combination allows for a more comprehensive understanding of samples by providing insights into size, molecular weight, and charge variation.

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With IEX, a change in salt concentration and/or pH of the mobile phase during the experiment is used to elute samples off a column with a charged stationary phase. For example, with a stationary phase of negatively charged beads and a mobile phase of increasing salt composition, negatively charged species will elute earlier. Once the salt composition has increased sufficiently, positively charged species will elute. Coupled with MALS, information such as molecular weight and radius can be measured while simultaneously gleaning additional charge information from IEX. This strategy can be particularly beneficial in cases where different sample species have the same or similar hydrodynamic volume but different net charges, enabling separation with IEX.

If you haven’t already set up HPLC CONNECT 4 and an ASTRA method for your system, doing so is easy! These topics are covered in this article: How do I use the HPLC CONNECT 4 software to control my Waters LC system? Open your ASTRA method within ASTRA via File | New | Experiment from Method.

Open an ASTRA Method file, select the Experiments icon, expand the Configuration option, and double-click the HPLC Instrument. This selection will display all of the information communicated to your Waters LC system to control its various modules, including your pump. In this window, click Edit Device Parameters to change parameters specific to your Waters LC system. In the pump method section, the pump time table can be changed to pull solutions of different salt compositions at different times during the method. In the example shown to the right, the salt content of the mobile phase has increased from the initial start to the end of the run.

When you have set up the composition profile for your IEX method, click OK to save these settings. Then, right-click the experiment name and select Save as Method to save this as a new method. Make sure you have set up your solvent reservoirs with the appropriate solutions! You are now ready for your IEX-MALS experiments.

Analysis within ASTRA for IEX-MALS experiments is similar to that for typical SEC-MALS experiments. Depending on your salt gradient, you may see a changing RI baseline through the run from your Optilab™ instrument. This is normal and expected, as the Optilab instrument will see changes in the concentration of your sample and the composition of the mobile phase. Run a blank injection, collect the data, and perform a Blank Baseline Subtraction and accounting for this shifting baseline. For more information, see our technical note TN on this topic.

If you regularly work with large solvent gradients, the Optilab High Concentration instrument may be better suited for your IEX-MALS needs. For example, the standard Optilab instrument can measure gradients between 0 – 450 mM NaCl without saturating, whereas the high concentration model can measure gradients up to 3.4 M NaCl.

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Support for Waters Arc™ Premier HPLC control, including setting up methods for ion-exchange chromatography, is only available in HPLC CONNECT 4 with ASTRA 8.2.1 and higher. Future releases will add support for more Waters LC systems. For more details see the ASTRA 8 User’s Guide or the HPLC CONNECT 4 User’s Guide.

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Top Features Readers Look for in Chromatography Columns

Learn the most common separation modes used by readers, and more from the latest chromatography columns survey results

The wide spectrum of columns available makes selecting this most important component of an LC system extremely difficult. Column choices span normal phase, reverse phase, size exclusion, ion exchange, hydrophobic interaction, and affinity chromatography. One is hard-pressed to find a more innovative, self-reflective instrument market.

Top 7 Questions You Should Ask When Buying Chromatography Columns

1. Based on your analyte(s), matrix, separation goals, and instrumentation, what column does the vendor recommend?
2. What benefits does this column offer over your current column? Performance, lifetime, reproducibility, etc.
3. How should you clean/prepare your sample prior to injection on the column?
4. How do you care for the column? Conditioning, cleaning, storage, etc.
5. What type of chromatographic media (fully porous, monolithic, core-shell) is going to provide the most benefit for your separation?
6. Do you need a unique selectivity (HILIC, polar endcapped, etc.) to separate any very polar and/ or nonpolar components in your mixture?
7. What column dimension is going to be most suitable for your loading requirement?

Separation modes used by survey respondents:

Reverse phase59%Normal phase39%Ion exchange26%Hydrophilic interaction (HILIC)23%Ion chromatography18%Affinity16%Chiral13%Gel filtration (GFC)13%Ion exclusion13%Gel permeation (GPC)11%Other10%

Column phases used by survey respondents:

C%Silica37%Anion exchange24%Phenyl24%Cation exchange21%C821%C18 (polar endcapped)19%Biphenyl13%Other13%Amino11%Cyano11%C48%PFP5%

Nearly 67% of respondents are engaged in purchasing new chromatography columns. The reasons for these purchases are as follows:

Require higher quality data 30% Increase column life 30% Addition to existing systems, increase capacity 28% Trying to reduce operating costs 25% Setting up a new lab / developing a brand new method 23% Require shorter run times / increased lab throughput 23% Working with more difficult samples that cause column clogging 20% Require more precise and accurate flow rates 19% Other 13% Reduce solvent usage and waste 11% Require a special size column 9% Reduce sample prep steps and time 6%

Top 10 features/factors respondents look for when purchasing chromatography columns:

Lot to lot reproducibility87%Technical performance74%Ruggedness / durability69%Initial purchase price of column53%Reputation of column manufacturer43%Applications support43%Column discount program42%Method validation / compliance support36%Specials and promotions34%Available applications literature26%

For more information on chromatography columns, including useful articles and a list of manufacturers, visit www.labmanager.com/hplc-columns

See results from previous surveys:

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