HPLC Tips & Tricks: Optimizing Injection Volume
Dr. Egidijus Machtejevas, Sr. Technical Advisor, Analytical Chromatography Workflows
Merck
The peak area has a linear relationship with injection volume/sample concentration if the column and/ or detector is not yet overloaded, and if the sample is dissolved in the mobile phase which flows through the column at the moment of injection (effects related to different sample diluents will be covered in one of the next HPLC tips & tricks). Commonly with sample load, we primarily associate the injection volume which, in HPLC, is usually determined by the autosampler setting. In the case of diluted samples, increasing sample volume can be considered as one option for improving detection limit/sensitivity. However, there are some effects related to increased injection volume and/ or concentration in HPLC/UHPLC which take place: the column can be overloaded if too much sample is injected onto the column; the height of the peak will increase with a larger injection volume; the peak width will broaden with an increase in injection volume. The sum of previously mentioned effects would mean that increasing injection volume can result in a decrease of resolution between the separated compounds (in case of overloading). It is important to keep in mind that changing injection volume, in general, would affect the peak (height, volume, and width) dependent on: the dimensions of the column (length and diameter), packing particle size and type (fully porous, superficially porous, or monolithic material as well as its surface area, functionalization density, chromatographic mode, and retention mechanism), and the peak retention (peak capacity value). Rule of thumb, for any chromatographic system change (shorter column, narrower column, smaller particle size, or shorter retention) that reduces the final volume of the peak eluting from the column, the volume of a sample should be proportionally reduced to avoid loss of resolution.
It is quite challenging to evaluate the sum of all these different parameters, therefore most often we turn to a more pragmatic approach and look only to column size and resolution minimum which is still acceptable. There is a generally accepted rule of thumb which recommends that the injection volume should be not more than 1%-2% of total column volume (considering a sample concentration of ~1 µg/µL, which is a standard value in analytical liquid chromatography); however, everyone could/should determine the actual maximal acceptable load by simply performing several different load injections and compare critical peak pair resolution or main peak chromatographic performance parameters. As an example, for the commonly used UHPLC column dimension of 50 x 2.1 mm which has 173 µL total volume, and a void volume of ~120 µL (considering a factor of 0.7 for a packing with fully porous particles) the injected volume should be in the range of 1.2 and 2.4 µL (when sample concentration is ~1 µg/µL), to limit band broadening and loss in resolution (few other examples: 4.6 mm I.D. and 50 to 250 mm length – ideal injection volume between 5.8 and 58 µL, 3 mm I.D. 50 to 150 mm length ideal injection volume between 2.5 and 14.8 µL. It is important to note that isocratic runs are much more prone to volume overloading effects than gradient methods. For isocratic runs, we can approximately calculate the recommended limit of injection volume by dividing peak retention volume (in µL, flow rate multiplied by peak elution time) by the square root of that peak efficiency (plates per column).
How might one see that the column is (volume) overloaded? When a very large volume of sample is injected into the HPLC column, the peaks begin to front more (peak symmetry factor < 1) and the retention time may decrease, resulting in a decline in column efficiency and separation resolution. The trick is to find the sweet spot, a balance between resolution and sensitivity (the sensitivity topic I already covered in issue 9 of this journal). Practically, it is easily done. Start with the smallest volume that your injector can reproducibly inject and keep doubling it until a max 3% of your column’s volume is reached. Then calculate the limit of detection and resolution dependence on injection volume. Be prepared for the need to accept a compromise between detection limit and resolution.
Other HPLC Tips & Tricks Articles (see also related articles)
For more information on HPLC method development download our guide A Practical Guide to HPLC - Method Development Guide or watch our webinar HPLC Method Development in 1.5 Days using the Selectivity Concept
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