Split or splitless: that is the question!

  • Figure 1:	Illustration of the gradient delay time from the point of mixing of the solvents to the head of column (y-axis) versus the flow rate (x-axis) in dependency of different gradient delay volumes.Figure 1: Illustration of the gradient delay time from the point of mixing of the solvents to the head of column (y-axis) versus the flow rate (x-axis) in dependency of different gradient delay volumes.
  • Figure 1:	Illustration of the gradient delay time from the point of mixing of the solvents to the head of column (y-axis) versus the flow rate (x-axis) in dependency of different gradient delay volumes.
  • Figure 2:	Analysis of 30 pharmaceuticals and personal care products on a YMC Triart C18 (50 x 0.3 mm, 1.9 µm) column using a gradient time of 12 s. Flow rate: 50 µL min-1, temperature: 50 °C, detection: triple-quadrupol mass spectrometer.
  • Figure 3:	Overlay of 100 consecutive gradients. For the sake of clarity, only every tenth gradient is shown. The dotted blue line represents the programmed gradient, while the solid lines represent the actual gradient delivered by the pump.

The development in the field of stationary phases needed new system designs of the established HPLC hardware. In particular, the introduction of sub-2 µm particles required an enlargement of the back pressure capabilities as well as the minimization of all system- and dead volumes. That is why the gain in efficiency by using smaller particles is closely related to the pump technology as we would like to demonstrate in the following.

Split or Splitless

An often described advantage of miniaturized separation techniques is the saving of expensive and toxic organic solvents. By using a 300 µm inner diameter (i.d.) instead of a 3.0 mm i.d. column, the solvent consumption at a given linear velocity can be reduced by the factor of 100. However, this advantage can only be utilized when a pump system without a flow-splitter is used. Not every system used as nano- or micro-LC can generate a “real” nano- or microliter flow rate. This means that for many systems the flow is splitted in front of the column to achieve the desired flow rate and thereby the overall solvent consumption cannot be reduced. That is why in the last years, mainly pneumatic syringe pumps are developed and used for this purpose. Another advantage of such systems is that high volume mixers are not necessary leading to extremely low gradient delay volumes which we would like to demonstrate in Figure 1. The time from the point of mixing the solvents to the head of the column is shown on the y-axis whereas the flow rate is plotted on the x-axis for different gradient delay volumes. If for example a UHPLC system with a gradient delay volume of 50 µL is used (red line in Figure 1) at a flow rate of 50 µL min-1, the resulting gradient delay time is 1 min until the gradient reaches the column. This time also needs to be considered for column re-equilibration after the solvent gradient.

Fast analysis

A gradient delay volume of 50 µL is quite low even for modern UHPLC systems. Often, the gradient delay volume is much higher (e. g.  200 µL) leading to higher gradient delay times.

Even though it is possible to use these systems for micro-LC when looking at the system specifications, the gradient delay volume is a decisive parameter in terms of the total analysis time. When the gradient delay volume is only 1 µL, which can be achieved for modern nano- and micro-LC systems, the gradient delay time for a flow rate of 50 µL min-1 is only 1.2 s. Therefore, very fast solvent gradients can be generated to achieve short analysis cycle times as is shown for the separation of selected pharmaceuticals in Figure 2. Detection was done using a triple-quad mass spectrometer with a fast data acquisition rate.

The analysis of all compounds can be accomplished within a few seconds using a gradient time of only 12 s. When using sub-2 µm particles, such fast separations can only be achieved at elevated temperatures (70 °C) when considering the maximum column back pressure of 600 bar. The linear velocity at a flow rate of 50 µL min-1 is about 1.7 cm s-1 assuming a column void time of 2.9 s. To operate a 2.1 mm i.d. column at the same linear velocity requires a flow rate of 2.45 mL min-1 which is out of the optimum range for the hyphenation to mass spectrometry with electrospray ionization (ESI-MS).

Applications

However, the question arises for which applications such fast separations are needed? Besides the high-throughput screening with hundreds or thousands of samples a day, this technology is ideally suited for process analysis to control and monitor chemical reactions. At this point, the miniaturization has an important advantage compared to conventional LC systems because the required sample volume can be reduced to the nanoliter range if needed. This is advantageous for processes which allow only minimally invasive analysis. Moreover, such fast gradients are suitable for comprehensive two-dimensional HPLC (LC x LC) to achieve cycle times of 30 s in the second dimension.

Another persistent prejudice of miniaturized pump systems is the lack of robustness. To refute this prejudice, Figure 3 shows an overlay of 100 consecutive gradients. For the sake of clarity only every tenth gradient is depicted.

The gradient delay volume was determined to be 0.93 µL, which means that very fast cycle times can be generated. Furthermore, it is evident that no deviations for the gradient response can be observed. This clearly underlines that the main worry of many users in terms of the reproducibility of micro-LC systems can clearly be rejected. In this context, Hetzel et al. have recently validated a method for the separation of antineoplastic drugs using micro-LC-MS/MS. The results clearly prove that the micro-LC-MS/MS hyphenation meets the requirements regarding the robustness in a routine laboratory.
 

Do you have problems with micro- or 2D-LC? Ask the experts at: adlichrom@iuta.de

Authors
T. Teutenberg1, T. Hetzel1, D. Loeker1, J. Leonhardt1

Affiliation
1Department Head Research Analysis
Institute for Energy and Environmental Technology e. V. (IUTA)
 

Contact
Dr. Thorsten Teutenberg

Department Head Research Analysis
Institute for Energy and Environmental Technology e. V. (IUTA)
Duisburg, Germany
adlichrom@iuta.de
 

Reference:

Hetzel et al., Micro-Liquid Chromatography Mass Spectrometry for the Analysis of Antineoplastic Drugs From Wipe Samples; Anal Bioanal Chem 408 (2016), 8221-8229) DOI:doi.org/10.1007/s00216-016-9932-y

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