LC Trouble Everywhere - - Chromatography Online
LC Trouble Everywhere

LCGC North America
Volume 30, Issue 1, pp. 36-41

Reference Standard Purity

Figure 1: Reconstructed chromatogram for a certificate of analysis. The arrow marks the column dead time.
In Turkey, a student complained that he had purchased a reference standard that was claimed to be >99% pure in the certificate of analysis, but when he ran it on his LC system, he found impurity peaks that greatly exceeded the <1% claim. He showed me a chromatogram that he obtained with a large analyte peak and a smaller impurity peak that indeed was more than 1% of the reference standard. He also had a copy of the certificate of analysis that contained a chromatogram showing a single peak that looked similar to the one I've recreated in Figure 1. The mobile phase was 90% acetonitrile, 10% water on a C18 column for the certificate of analysis; unfortunately, I did not note the conditions for his analysis.

However, even with this limited amount of information, it is possible to make an educated guess about the source of the problem. If you recall from previous installments of this column, I've stated many times that the ideal isocratic chromatogram will have peaks in the 2 < k < 10 range, or if this is not possible, 1 < k < 20. This gives the analytes sufficient time to interact with the column to achieve "good" chromatography, yet keeps the run time from being excessive. Normally, we calculate the retention factor, k as

k = (t Rt 0)/t 0                   [6]

where t R and t 0 are the retention time and column dead time as noted in equation 3. I did not include a time axis in Figure 1, because I can't remember what it was in the certificate of analysis, but the units in equation 6 cancel, so we can use a ruler to calculate k from the chromatogram. The first disturbance in the baseline (arrow in Figure 1) marks t 0 and we can use the top of the peak as t R to calculate k ≈ 0.3. This is much less than the desired minimum k = 2. When peaks are eluted with k << 1, there is little time for the analyte to interact with the column, and the chances of having unresolved peaks present is increased. This is especially important when using a chromatogram to certify peak purity. The small tail on the peak may be peak tailing that is normal with many peaks or it may be a subtle indication that an impurity is present. There is no excuse for producing a chromatogram like this for a certificate of analysis, except laziness, ignorance, or impatience. It would be easy to increase k for the peak from 0.3 to 2 < k < 10 by changing to a weaker mobile phase, such as 70% acetonitrile, 30% water. At that point, a more convincing case could be made to show that the peak was indeed >99% pure. Although I don't remember the conditions of the user's chromatogram, I do remember that the analyte peak was significantly broader than that in the certificate of analysis, suggesting that the retention time was much larger. My answer to the question was that the certificate of analysis didn't convince me of the purity of the reference standard, so I would be more likely to trust the analyst's chromatogram in which k >> 0.3.


It doesn't matter where we live; LC problems have no international boundaries. In fact, I've noticed that chromatographic terminology often is adopted directly into many different languages. In some ways, chromatography really is a universal language. It reminds me of a time when a visitor from one of the Asian countries visited my son's classroom when my son was about 12 years old. The visitor asked if the kids wanted to learn some of his language. All were excited about the prospect. The visitor carefully recited, "computer," "Coca-Cola," and "McDonalds." Maybe he should have included "chromatography."

John W. Dolan
John W. Dolan "LC Troubleshooting" Editor John Dolan has been writing "LC Troubleshooting" for LCGC for more than 25 years. One of the industry's most respected professionals, John is currently the Vice President of and a principal instructor for LC Resources in Walnut Creek, California. He is also a member of LCGC's editorial advisory board. Direct correspondence about this column via e-mail to


L.R. Snyder, J.J. Kirkland, and J.W. Dolan, Introduction to Modern Liquid Chromatography, 3rd Ed. (Wiley, Hoboken, New Jersey, 2010), p. 120.


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