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Chromatography Today Help Desk Impact of flow rate on retention time
Tony Edge, School of Chemistry, University of Liverpool, Crown St, Liverpool L69 7ZD
There are on occasion times when there is no obvious reason for the experimental arrangement and so it was when a colleague of mine, David Dunthorne, asked if it was necessary to use trifluoro acetic acid (TFA) in the determination of the chromatographic efficiency of a new column. Looking briefly at the compounds, ketoprofen and uracil, there did not appear to be a rationale as to why TFA should be used and on this basis TFA was replaced with formic acid, since the method was eventually going to be run on a mass spectrometer, where TFA can be quite detrimental to the detector response [1]. It was also felt that some of the UV detector ripple effects that can occur with the use of TFA [2] may be eliminated if the assay was moved to formic acid. The detector issues are associated with the relative high UV absorbance that TFA has compared to the mobile phase (ACN and water in this case) and the resulting sensitivity associated with the mixing process of the mobile phases.
The tests to determine the chromatographic efficiency are trivial and involve the sequential injection of an injection marker (uracil) and a retained compound (ketoprofen), at different flow rates. The mobile phase used was 0.1%TFA in 25:75 ACN:Water with the column thermostatted at 40°C. The data that is typically plotted is the flow rate or linear velocity of the mobile phase versus the chromatographic efficiency in the form of Height Equivalent to Theoretical Plates (HETP), which is a measure of the peak width.
The generated plots which were done on a range of solid core particles looked good and were in agreement with the expected results. However, it was noted that the retention factor varied with the flow rate, from 12 down to nearly 8. This is quite a significant impact on the retention factor and so further investigation was needed to determine the rationale behind the relative shift in retention times between the two compounds.
Where;
c is a factor which relates to the retention mechanism and is generally assumed to be independent of the flow rate.
tr
is the retention time of the analyte, in this case ketoprofen Q is the volumetric flow rate V0
is the volume of the chromatographic system between the injector and the detector.
Although retention time is often used to determine the time spent on the column a better variable to use is the retention factor, which is defined as.
The retention factor should not vary with flow rate since the equation uses the t0
value, which is associated with the retention of an unretained peak, uracil, and would compensate for the increase in flow rate.
The retention factor data is shown in Figure 1, and it can be clearly seen that the retention factor does vary with the flow rate.
Where L is the length of the column and N is defined as; Figure 1. Effect of flow rate on the retention factor for ketoprofen. tr is the retention time of ketoprofen
is the peak width at half the height for ketoprofen The equation defining the retention time can be written as;
w0.5
As part of the investigation to determine the cause, the retention time for both compounds was plotted as a function of the flow rate and it could be seen that for both compounds a linear relationship was established between the flow rate and the reciprocal of the retention, and indeed the r2
value was very close to one, suggesting
that the data was very good. The issues associated with the use of r2 was covered in a prior Helpdesk article, and this is probably a prime
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