Abstract
The open access LC/MS has become particularly popular in determining final chemical reaction-product purity and integrity, as well as to monitor chemical reactions. However, the open access environment suffers from clogging since chemists need to run thousands of different samples, which have varying solubility levels. Several practices to reduce or prevent clogging have been trialed in the open access environment, including filtering of samples and increasing sample solubility. However, these methods have generally failed to solve the persistent clogging problem, costing the operators money in terms of purchasing replacement parts due to the effects of clogging. In addition, failures due to clogging haves resulted in significant investment losses in terms of repairs and man-hours. This paper presents a novel technique for the prevention of clogging in open access LC/MS through back-flushing of the injector seat. It was postulated that back-flushing the instrument’s injector seat can increase the instrument’s tolerance to an increase in sample load, thus reducing clogging. This hypothesis was tested using a modified prototype instrument. The results showed that back-flushing the injector seat reduced incidence of clogging even when loading a sample at 1000 times the normal particulate levels, which, In turn, reduced time and resources spent on repairs and maintenance.
1. Introduction
Open access liquid chromatography, including liquid chromatography/mass spectrometry and flow injection analysis, has become an increasingly important tool for the monitoring of chemical reaction progress, as well as to assess the purity and/or integrity of resulting chemical entities [1]. In this case, open access LC/MS allows chemists to carry out parallel chemical synthesis reactions. However, this trend has also resulted in a major challenge for chemists conducting high pressure liquid chromatography with regard to clogging, a problem that has long troubled users of various chromatographic techniques. Indeed, clogging is a particularly salient issue in the open access environment since chemists need to run thousands of different samples with widely varying solubility levels.
Several practices have been several implemented over time to prevent or mitigate the effects of clogging, while maintaining the physical separation and mass analysis capabilities of the technique. Nevertheless, these practices have not been entirely successful. For instance, one technique uses a liquefied sample normally with low solid levels and low particulate contaminant levels to prevent clogging while maintaining highly efficient and reproducible solid phase extraction [2]. Another technique to prevent or reduce clogging involves the filtering of test samples specifically to remove any particulates in the sample that might cause clogging. Additionally, open access LC/MS users have also attempted to reduce clogging by changing the liquid chromatography gradient with the aim of accommodating samples that are not soluble in water. Moreover, users have also attempted to solve the issue of clogging by increasing sample solubility, which is achieved by increasing the sample temperature to enhance interactions between the solid and liquid phases [2]. Users have also attempted to protect the column from the effects of clogging by adding an inline filter to the column. Finally, users have also attempted to reduce clogging through the use of supercritical fluid chromatography-mass spectrometry for suitable samples.
In spite of these attempts to reduce clogging in open access LC/MS, the clogging problem remains unresolved, increasing maintenance and repair costs for operators in terms of time and resources due to downtime, replacement parts, and staff hours spent. Essentially, the open access team is tasked with ensuring that the LC/MS instruments are hard to break and easy for operators to use [3]. For example, in our operations, around fifty thousand samples were run through the open access LC/MS in a year, from which the operators had to solve issues such as sample, software, and hardware failures. More than half of these failures were as a result of clogging as evidenced in table 1. The main cause of clogging as identified in this case was samples with diverse physio-chemical properties, such as solubility [3].
2. Material and Method
2.1 Chemicals
All solvents were of HPLC Chromasolv grade, from Sigma Aldrich (St. Louis, Missouri). Water was purified by a Millipore MilliQ Gradient (Billerica, Massachusetts).
2.2 LC-MS
All data was gathered on an Agilent 1260 LC with MSD Agilent model 6120 single-quadrupole mass spec detector with atmospheric spray ionization source. The LC instrument includes a binary pump Agilent model G1312B with upper pressure limit of 600bar attached to auto-sampler Agilent model G1329B which uses external try for sample submission. The column compartment Agilent model G1316C which is attached to diode array Agilent model G1315C. The instrument acquisition and data handling was done with ChemStation rev. B.05.01. The sample submitted using Agilent Easy Access rev. A.06.
2.3 Elution Conditions:
The general open access method consist of 2min and 3min gradient of 5-95 %B in 1.5 and 2.5 min, with 0.5 min hold T 95%B. Solvent A: Water (0.1% formic acid + 0.05% ammonium formate). Solvent B: Acetonitrile (5%H2O+0.1% formic acid+0.05% ammonium formate). The flow rate is 1.2 mL/min. The Column: Waters Acquity BEH C18, 2.1x30mm, 1.7µm particle size; Column Temperature 80 °C
2.3 Back-Flush Scheme:
The back-flushing unit consists of stainless steel filter Agilent part 5067-4638, a stainless steel tee Valco part ZT1C and customized waste tray for injector without washing port like Agilent model G1329B. Figure 1 shows flow path of injector and Figure 2 flow path of injector after back-flush modified. This setup prevent solid particulate matter interning into the system by filtering them out at needle seat, where most particulate introduce to system Figure 2b. In next injection during sample aspiration this debris is removed from system by back-flushing the needle seat Figure 2a. This automated self cleaning after each injection does not need any method alteration, injection programming or valve programming.
3. Results and Discussion
To reduce the costs of replacing the open access LC/MS instruments as a result of clogging, a novel but inexpensive plumbing technique was tested on an open access LC/MS prototype. In this case, it is postulated that back-flushing the instrument’s injector seat, through which the operators introduce particulates into the system, can increase the instrument’s tolerance for an increase in sample load thus reducing clogging [3]. The modified plumbing scheme, as shown in form of a schematic in figure 1, increased the amount of particulate that can be loaded into the injector seat. As such, it was further postulated that the operators using this back-flushing technique can inject up to twenty times the normal levels of particulate without the inconvenience of clogging. On the other hand, it was found that injecting a similar sample into the injector seat of a system without the back-flushing modifications resulted in the instrument clogging almost immediately [3]. From the testing process, it emerged that back-flushing the injector seat allows the operator to avoid clogging even while loading a sample at 1000 times the normal particulate levels.
While the idea was initially received by vendors with some skepticism, we showed that the prototype back-flushing system could withstand twenty times the concentration of a standard sample into the injector seat with no clogging. On the other hand, a similar sample placed in the injector seat of an unmodified system resulted in immediate clogging [4]. Therefore, the different outcomes between the experimental prototype instrument and the control instrument proved the hypothesis that back-flushing significantly reduces clogging in the open access LC/MS instrument. Furthermore, when super-saturated bi-phasic test samples were loaded into the injector seat, it was demonstrated that the novel scheme was able to handle at least 103 times of a normal sample load without clogging [4]. In addition, back-flushing modification was also installed in a heavily used open access LC/MS system, in which at least 51 of 96 service calls in 2014 had to do with clogging that led to shutdown of the system. However, following installation of a back-flushing system in the instrument’s injector seat, the system has not undergone any shutdown as a result of clogging as seen in figure 3.
Afterwards, a new scheme was also installed for another four open access LC/MS instruments, following which the clogging was found to reduce significantly. The modification process was faced by one significant challenge related to the customization of the tray to ensure it was compatible with a wide array of solvents. This is an important capability for an open access LC/MS machine, which has to load close to 50,000 diverse samples every year [4]. The initial solution, which involved the gluing together of a makeshift tray using existing parts, failed due to low durability of the resulting tray. A second solution involved the use of injection-mold plastic to make the injection tray. However, this solution also proved challenging due to the high timelines and costs estimated at more than $1,200 per unit. Eventually, the study identified a less costly method using 3D technology, shown in figure 4, to design a custom-made tray, which allowed for the testing of different designs that enabled for the fine-tuning of the instrument’s specifications [4]. The latter technology had a fast turn-around time, while it was also low-cost at only $10 per unit.
The back-flushing method has proved successful, with no clogs reported in the system since the modifications were installed. At present, the team has made more modifications to a further three systems, with the results showing a significant reduction in the number of clogging reports as seen in figure 5. No clogging was reported as a result of sample loading for many months after installation of back-flush. As more instruments are modified with this novel technique, it is expected that the costs of clogging in terms of downtime and resources will be significantly reduced
The other advantage of this back-flush set up is that the tip of the needle is rinsed after each injection by the leftover liquid from back-flushing sitting on the needle seat see Fig.4.b. This allowed us to cut the needle wash step and shorten the injection to injection cycle time by 30 seconds. This increased the sample through put efficiency by10%. Every week, we replaced these wash vials with clean solvents. This manual labor is eliminated and the system made more self sustainable. It increases the self sustainability of the LC/MS and reduced the steps we had to carry out. Notably, this setup does not influence the system’s chromatographic performance because the back-flushing mechanisms are placed upstream of the chromatography column see Figure 6. In this case, the back-flushing system does not need any significant alterations to the existing LC/MS methodology and instrument. Further, the back-flushing modifications do not affect the system negatively in relation to carryover and precision.
4. Conclusions
The back-flushing method has proved successful, with no clogs reported in the system since the modifications were installed. At present, the team has made more modifications to a further three system, with the results showing a significant reduction in the number of clogging reports. No clogging was reported as a result of sample loading for the month of August and, as more instruments are modified with this novel technique, it is expected that the costs of clogging in terms of downtime and resources will be significantly reduced.
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