ion-chromatographyIon chromatography (IC) is a well-established liquid chromatographic technique which is increasingly used for pharmaceutical analysis. Applications of IC in the pharmaceutical industry include drug assay, assay of impurities in drugs, counterion determination, and measurement of drug product excipients. IC is appropriate for pharmaceutical identification tests (USP <191>), and is the prescribed technique for the determination of citrate, phosphate (USP <345>) and is a proposed technique for zinc determination (USP <591>) in drug products. I learned all of this in one of my vlog episodes earlier this year!

In October I seized the opportunity to co-ordinate a survey of the pharma industry followed by a live expert panel discussion exploring the role of IC in the USP monograph modernization initiative, and applications of IC in the modern pharmaceutical laboratory.


Meet the Panellists: Leonel Santos, Ph.D. Director of Chemical Medicines, U.S. Pharmacopeial Convention; Jeffrey Rohrer, Ph.D. Director of Applications Development, Thermo Fisher Scientific; Edith Chang, Ph.D. Scientific Liaison, U.S. Pharmacopeial Convention; Shreekant Karmarkar, Ph.D. Director, Program Management, Baxter Healthcare and Michael Chang, Ph.D. Scientific Liaison, U.S. Pharmacopeial Convention


The results of the survey can be seen summarised in this handy infographic:


The live panel discussion had an extended Q & A session, but as always with such hot topics, not every question could be answered in the time allotted…so, I caught up with our in-house IC guru, Dr Jeffrey Rohrer, to get answers to some of the most frequently asked questions. These were far too good not to share, and since it is the time of year for giving, I present to you the gift of IC!

Q: How can IC be more advantageous than other chromatographic methods for determination of pharmaceuticals?

A: When the analyte is ionic at pH 7, IC should always be considered for the analysis (please note that if the analyte is an ion at another pH there may also be an IC approach available). While other chromatography techniques can sometimes analyse ionic molecules, there are often compromises in terms of resolution or ruggedness.  Ion chromatography is ideal for ionic analytes and usually the mobile phase requires lower cost, in terms of purchase and especially disposal, of organic solvents. Additionally, the mobile phase can often be automatically generated which increases productivity and significantly reduces error.

Q: How do you compare IC methods against the manual measurements such as turbidimetry, TLC or titration? How is IC superior compared to AAS or ICP, for example?

A: There are certainly many ways to compare. For example, you could compare the labour of each for a given number of samples, you could compare precision of each for a set of samples, and with a standard reference material that you could then compare the accuracy of each method.  It is important to remember that one IC assay could determine multiple analytes while other methods are unlikely to do that. To the second question, IC is not necessarily superior to those techniques that are typically applied for determining cations or metals.  As mentioned earlier, IC can determine multiple cations in the same injection, and it can determine another analyte sometimes required in pharmaceutical analysis, ammonia (as ammonium), in that same injection.


Q: How can we make IC more robust for release testing in QC?

A: There are numerous ways to accomplish this.  First, evaluate your separation:

  1. Are there closely eluting peaks?
  2. Are unusual mobile phase conditions required for that separation (i.e. a step change of eluent concentration that is vital for resolution)?

If the answer is yes to either question it is prudent to evaluate at least one other column from a different lot to see if you can accomplish the desired separation.  You should also evaluate where the chromatographic values reported on the quality assurance report (QAR) fall in the range of specifications for that column. Other ways to make your method more robust is to control column temperature, control cell temperature, use full loop injection, evaluate deliberate 10% changes in chromatographic parameters to determine how they affect your separation, and use eluent generation to prepare the mobile phase.

Q: Do you have any tips to obtain sensitive, robust and reproducible results, considering long-term use specifically (e.g. during stability studies over 5 years)?

A: First you must design a method where you have good resolution of your analyte or analytes of interest from other compounds. This will prevent your analysis from being affected by natural changes in the column as it ages with use (i.e. loss of capacity) or normal manufacturing variations. In terms of sensitivity you do not want to be routinely measuring a peak that is < 10X signal to noise (S/N). If you are measuring a peak <10X S/N you will have variation just due to natural variations and random error. It is important to track common chromatographic parameters (background, noise, resolution, backpressure) to see how they vary and how their variation affects your results.  If your method must have closely eluting peaks you should evaluate more than one column and the columns should be from different lots to make sure the separation is possible with natural column manufacturing variations. Testing your method according to USP General Chapter <1225> Validation of Compendial Procedures will give you some indication of method ruggedness.

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Q: How can specificity be proven using conductivity detection?

A: The nature of the conductivity detection is that it is specific for charged compounds, which makes it rather non selective. When we are doing anion analysis the retained peaks are anionic, and with cation analysis the retained peaks are cationic. The good news is that in pharmaceutical analysis we generally know what we are analysing, and what the potential impurities are. Therefore in most analyses we know what compound to expect. With standards for those compounds we look for matching retention times in our sample analysis and we can spike the sample with the standard to demonstrate coelution.  Putting a mass spectrometer after the conductivity detector can also help to confirm specificity.


Q: Many non-chromphoric compounds, like amino acids, are analysed using ion pairing in HPLC and derivatization, even though they can be analysed easily using ion chromatography with PAD. What are the advantages of the latter approach?

A: The advantage of analysing amino acids and non-chromophoric compounds by IC with PAD is that it provides a sensitive determination without encountering time, cost, and possible erroneous sample derivatization limitations- such as incomplete or differential derivatization. IC-PAD is a direct, rather than indirect, determination of the analyte.

Q: What are your recommendations for the analysis of phosphate containing molecules by IC-MS?

A: Phosphate, polyphosphates, nucleotides, and sugar phosphates, including inositol phosphates can all be separated by IC, typically with a sodium or potassium hydroxide mobile phase. The suppressor used for suppressed conductivity detection makes the effluent from the conductivity cell amenable for negative mode electrospray ionization and subsequent MS detection. Any phosphate containing compound that can be separated with a mobile phase that can be suppressed will be available for MS detection.

Q: When proposing a method for the USP or EP, do you start with the simplest IC method and work your way up? For example, single column IC (SCIC) is much easier to use than suppressor-based IC. If the method is suitable for SCIC, would that automatically be proposed?

A: Every method should be designed as fit for purpose. Therefore if a single column (no suppressor) will meet the needs of the method in terms of reproducibility, sensitivity, and ruggedness, it would be appropriate. Simple is certainly better if it delivers the required reproducibility, sensitivity, and ruggedness. In general I would start with something that has already been done (i.e. do a good literature search to start).  From there I would see if that method can be either used as is, adapted for my needs, or  improved upon with a new column (perhaps to achieve better resolution and/or faster analysis) or some other advancement in the technology (e.g. eluent generation to improve reproducibility).

If you have any more questions that you’d like answering on this topic please do enter them in the comments below!

To learn more about the application of ion chromatography for pharmaceuticals and biopharmaceuticals visit:

To request more information on our pharmaceutical analytical solutions visit:

Check out our brochure on ion chromatography for pharma & biopharma.

Watch this video of Dr Jeffrey Rohrer Talking Ion Chromatography for Pharmaceutical Analysis.

Watch the panel discussion webinar NOW on-demand.

Visit our Ion Chromatography & Pharmacopeial Monograph Modernization microsite with Separation Science.

View the infographic on the usage of ion chromatography in the bio/pharmaceutical industry.

Watch BioPharMoore Episode 9: Modern IC, the USP and Me.