34 February / March 2021
Considerations for Sample Injection in High-Throughput Liquid Chromatography
James P. Grinias1,* 1
2 and Edward G. Franklin2
Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ USA Regis Technologies, Morton Grove, IL, USA
* Corresponding Author:
grinias@rowan.edu Introduction
The implementation of ultrafast liquid chromatography techniques to reduce method time and increase analytical throughput has grown tremendously in recent years. Instruments capable of higher pressures and flow rates, coupled with modern column technologies that can provide sufficient chromatographic efficiencies even at very short lengths, have provided the means to drastically increase separation speeds. Improvements to detector modules and new data processing algorithms are enabling effective signal acquisition at the requisite rates. The rate of autosampler operation has also increased, but has lagged behind these other advancements and now represents one of the primary bottlenecks for further increases to sample throughput in LC [1]. In this overview, current injector and autosampler technology will be discussed, along with recent strategies that have been used to reduce injection cycle time and how these might be further developed in coming years.
Autosampler Operation and Injection
In a typical method sequence that is set up within a chromatography data system (CDS), a series of runs are listed that detail which vial is to be sampled and which chromatographic method is to be used for each injection. In a standard instrument operating mode, the autosampler sequence occurs before each injection. This sequence typically includes movement of the needle to the designated sample vial position, a sample draw, movement back to the injector valve (if a needle seat port is used), and a series of needle washes to reduce sample carryover [2, 3]. This approach has multiple limitations that can increase instrument cycle time and thus reduce overall analytical throughput. The primary issue is the fact that the autosampler sequence and the chromatographic run are performed asynchronously, which means that the total instrument cycle time is the sum of both operation times rather than simply the slower of the two. The simple fix to this limitation is to simultaneously perform the autosampler sequence with the preceding chromatographic run so that as soon as one chromatographic analysis is finished,
Figure 1. Fifteen consecutive injections of a 20 s cycle time separation of acetaminophen (peak 1), aspirin (peak 2), benzoic acid (peak 3), and salicylic acid (peak 4) over 5 min coupled on a single chromatogram (from fifteen individually collected chromatograms) with vertical dashed lines designating each injection point. Adapted with permission from [7].
the next can begin immediately because the sample has already been delivered to the injector. Such functionality is often available as a setting of modern CDSs, using functions such as ‘PrepareNextInjection’ [4], ‘Enable Overlapped Injection’ [5], or “Prep Ahead” [6] within the autosampler control code. This approach was demonstrated for the analysis of over-the-counter (OTC) analgesic compounds with a complete
chromatographic cycle time of 20 s (total 5 min analysis time for 15 replicate runs) [7]. The short separation time was achieved by operating a 2.1 x 50 mm column packed with sub-3 µm core-shell particles at 1.3 mL/min, which required UHPLC instrumentation as the column pressure exceeded 750 bar. The autosampler sequence time was reduced to less than 13 s by limiting the time of the
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