Evaporation is a process used to concentrate samples by vaporizing and removing liquid solvents, leaving behind the dissolved solid components. Evaporation is essential for almost all analytical chemistry laboratories, and its primary functions include a) concentrating analytes to match the instrument’s detection range, b) changing the solvent system to adapt to instrumentation requirements, and c) removing reagents or components that could interfere with the analysis. Scientists seek an evaporation process that is straightforward, rapid, and reliable, ensuring no loss, alteration, or contamination of the analyte. Two major types of evaporators are used in analytical testing labs: vacuum concentrators and nitrogen blowdown evaporators.
The rotary and centrifuge evaporators fall into the category of vacuum evaporators, while the TurboVap® series belongs to nitrogen blowdown evaporators. Unlike vacuum evaporators, which require a vacuum pump, heating system, and cooling system to facilitate evaporation and condensation, the nitrogen blowdown evaporator only requires a consistent flow of clean, dry nitrogen with proper temperature control, which makes them simpler, more cost-effective, and easier to maintain. Nitrogen blowdown evaporation is highly effective in drying small sample volumes. This makes it an excellent choice for analytical laboratories conducting high-throughput assays or any experiments that require the removal of excess solvents during sample preparation.
Nitrogen blowdown evaporation relies on two critical factors: temperature and nitrogen flow rate. Temperature plays a significant role in the evaporation process. Higher temperatures increase the kinetic energy of molecules on the liquid surface, enabling them to overcome intermolecular forces and transition into the vapor phase more rapidly.
Nitrogen blowdown evaporation works by continuously blowing nitrogen streams onto the liquid surface. This action displaces the air that carries the vapor, lowering vapor pressure and accelerating the conversion of solvent molecules from liquid to gas. Higher nitrogen flow rates result in more solvent molecules entering the vapor phase, thereby shortening the drying time. The proximity of the nitrogen flow to the liquid surface also affects vapor clearance efficiency.
For optimal efficiency, the temperature should be set close to the solvent's boiling point, while the nitrogen flow should be as close to the liquid surface as possible. However, using tempera- tures near the boiling point can be impractical, as excessive heat may cause analyte loss, oxidation, or degradation. Also, high nitrogen flow rates with close gas-liquid distance can result in liquid splashes, cross-contamination, and sample loss.
Although nitrogen blowdown evaporation may seem straight- forward, it is crucial to fine-tune conditions to ensure accurate control of all parameters affecting evaporation performance. This ensures that the analytes remain unaffected by the drying process, providing consistent results across different samples and batches.
Nitrogen blowdown evaporation is versatile and can be used for a wide range of solvents. This includes volatile solvents like pentane, methanol (MeOH), dichloromethane (DCM), ethyl acetate (EtOAc), hexane, tetrahydrofuran (THF), and methyl tert- butyl ether (MTBE). It is also effective for semi- or low-volatile solvents such as water, acetonitrile (ACN), pyridine, 2-propanol, and methanol-water mixtures.
Drying volatile solvents is generally faster and requires lower temperatures and less nitrogen compared to drying the same volume of low-volatile solvents. However, with optimal evaporation conditions, nitrogen blowdown evaporation can achieve excellent performance and efficiency for both semi-volatile or low-volatile solvents.
Evaporation is a crucial component of high-throughput sample preparation methods. In most analytical testing laboratories, samples are processed with 24-, 48-, or 96-well microplates, necessitating a concentration system compatible with these formats. TurboVap® 96 Dual (Figure 1) is an automated nitrogen blowdown solvent concentrator specifically designed for well plates. It can simultaneously process two well plates, allowing for either dual or single evaporation within the 96-well plate format, as well as other formats such as 48- or 24-well plates and 48 vials. The TurboVap® 96 Dual builds on the solid foundation of the historic TurboVap® product line known for its reliability and performance. In this section, we detail the key experimental factors critical to the well-plate evaporation process and explain how TurboVap® 96 Dual manages these factors to achieve high performance.
Continuous nitrogen flow disrupts the vapor layer above the liquid surface, facilitating the molecular transformation from liquid to gas. Precise control of gas flow is crucial for effective evaporation. From a physical chemistry perspective, increasing nitrogen gas flow can significantly shorten the evaporation time, especially for low-volatile solvents. However, strong gas flow may cause liquid splashes and blow the sample away, leading to low recovery and the risk of cross-contamination. These issues are commonly encountered in well-plate evaporation, especially when the samples are filled near maximum volume capacity.
In previous versions of nitrogen blowdown evaporators, users had to monitor the evaporation process closely and adjust the gas flow to maintain efficiency and prevent cross-contamination. These manual operations are difficult to replicate and scale, causing variations within and between plates.
The TurboVap® 96 Dual offers direct control over static gas flow rates, ranging from 25 to 90 L/min. The system also supports stepwise or linear gas flow gradients. To maximize efficiency, users can program a method that allows the instrument to automatically control the flow rate over time during the evaporation process. For example, the gas flow rate can start low at the beginning to prevent liquid splashes and then gradually increase to mid- or high levels as the liquid volume decreases, thereby accelerating the overall evaporation process.
Temperature is a key factor in the evaporation process. It can be controlled by directly heating the solvent, the environment, and/or both. Ideally, maintaining a temperature close to the solution’s boiling point can accelerate evaporation. However, considering the thermal stability of analytes/samples in bioanalysis, the typical evaporation temperatures range between 40-80 °C. Heat-sensitive samples should be dried at lower temperatures (30 to 40 °C) with adjustments to gas flow and gas-liquid distance to compensate for evaporation efficiency.
The TurboVap® 96 Dual provides precise temperature controls over the microwell plate and nitrogen gas that is blown onto the liquid. The temperatures of these two sources can be set to the same or different values. Additionally, it supports defining stepwise or linear temperature gradients over time for both heating sources, enhancing the flexibility and efficiency of the evaporation process.
Sample proximity to the flow source refers to the distance between the nitrogen supply needle and the liquid surface in the well plate. Theoretically, the smaller the gas-liquid distance, the better the evaporation efficiency. However, to prevent liquid splashes and cross-contamination, the gas-liquid distance should always be maintained at 1-3 cm, depending on the gas flow and solvent viscosity. For example, the gas-liquid distance for water can be shorter than DCM, due to water’s higher surface tension, which reduces the likelihood of splashing and creeping into the neighboring wells.
In the TurboVap® 96 Dual, the gas-liquid distance is controlled by setting the plate height. The optimal plate height setting can vary depending on the type of plate and the volume of solution being evaporated. Therefore, it is recommended that users check the plate height settings in advance using an empty plate marked with the estimated liquid surface before starting the experiment.
Similar to temperature and gas flow control, the TurboVap® 96 Dual allows users to set either a static plate height for the whole evaporation process or a plate height gradient (stepwise or linear). This feature helps maintain an optimal gas-liquid distance (e.g., 1-3 cm), improving nitrogen efficiency and reducing evaporation time.
The properties of solvents, including their physiochemical characteristics, composition, and volume, significantly influence the evaporation time.
Volatile to highly volatile solvents can typically be evaporated in approximately 15 minutes at temperatures between 30-40 °C. However, low-volatile solvents, especially those containing water, acids, bases or salts, may require several hours to evaporate under mild temperatures. One of the most effective ways to expedite the evaporation of low-volatile solvents is to increase the temperature to 60 °C or higher, provided the analytes or samples are heat-stable. Alternatively, applying a high gas flow rate (e.g., 60-90 L/min) can also speed up the process. By optimizing the gas flow and gas-liquid distance (plate height), it is possible to achieve evaporation efficiency without exposing the samples or analytes to exces- sive heat. This approach is particularly beneficial for drying heat-sensitive analytes from low-volatile solvents.
Nonspecific binding and extractable/leachable interferences are common challenges in LC-MS and GC-MS analysis. Nonspecific binding occurs when analytes are adsorbed onto the plate material, making complete recovery with a solvent impossible. Extractable and leachable interferences involve unwanted molecules that are extracted from the plate that interfere with analyte detection or lead to misinterpretation of the results.
While various factors can contribute to these issues, one of the most significant causes is the plastic materials used to manufacture plates. Therefore, it is crucial to evaluate the source of well plates (or tubes) that come in contact with the sample solutions, especially those used in the evaporation process.
As a leader in sample preparation and separation science, Biotage® understands the importance of material integrity for analytical testing performance. To ensure the reliability of plates in the analytical workflow, Biotage® selects only the most stable and low-binding plastics, accompanied by strict quality control throughout the manufacturing process.
Evaporation is a gas-liquid equilibrium process where the solvent molecules move between vapor and liquid surfaces. Beyond gas flow and temperature, which directly affect the sample, the surrounding environment, including room temperature, airflow, and humidity, also impacts the consistency and reproducibility of evaporation.
When evaporating a sample plate in an open environment (e.g., inside the ventilation hood), even with the same nitrogen flow and temperature, samples in the central wells evaporate more quickly than those in the peripheral wells. The actual temperatures in different wells on the same plate can vary slightly, despite being heated at the same temperature. These differences cause variations in evaporation and, ultimately, in analytical results.
To mitigate the impact of the surrounding environment, the TurboVap® 96 Dual features a fume hood design to isolate the evaporation process from the surrounding environment. It includes two independent chambers, which can operate under the same or different evaporation conditions. The fully insulated chamber allows precise control of evaporation conditions across the plates, ensuring highly reproducible evaporation performance within and between plates. Additionally, each chamber has its own access to the ventilation system, signifi- cantly reducing the risk of chemical exposure to personnel. These lab safety features enable the TurboVap® 96 Dual to be used as bench equipment without the need to place it inside the ventilation hood.
It is also important to consider unmodifiable environmental factors, such as altitude. For example, the time required to evaporate the same volume of water will differ between labs in Denver, Colorado, and Charlotte, North Carolina, even if all other conditions are perfectly controlled.
The TurboVap® 96 Dual system offers three operation modes:
Manual mode: Allows direct control of static evaporation parameters, including gas flow, temperature (gas and plate), and plate height, without a time limit.
Time mode: The system automatically executes the set static conditions for a specified time period.
Method mode: Enables users to specify a ramp and save parameters such as gas flow, rate, temperature, and time. Once established, the method can be easily applied, allowing the system to precisely execute the programmed parameters for optimal efficiency and reproducibility.
Evaporation method development relies on understanding the evaporation behavior of solvents. For example, knowing the time required to evaporate 1 ml of a specific solvent or solvent mixture at specified temperatures and liquid-gas distances (plate heights) provides a foundation for estimating starting conditions and time needed for developing the actual evaporation method. Figures 2 and 3 demonstrate the influence of temperature, gas flow, and plate height on the evaporation performance of several solvent systems serving as a starting point for developing evaporation methods.
|
Conditions |
Low flow |
Mid flow |
High flow |
|||
|
Step |
1 |
2 |
1 |
2 |
1 |
2 |
|
Gas Flow (L/Min) |
25 |
25 |
50 |
50 |
90 |
90 |
|
Gas Temp (°C) |
40 |
40 |
60 |
60 |
40 |
40 |
|
Plate Temp (°C) |
40 |
40 |
40 |
40 |
60 |
60 |
|
Plate Height (mm) |
55 |
75 |
55 |
75 |
55 |
75 |
Figure 2. Evaporation time for semi- or low volatile solvents under low-, mid -and high- N2 flowrates.
|
Conditions |
Static plate height |
Gradient plate height |
|
|
Step |
1 |
1 |
2 |
|
Gas Flow (L/Min) |
50 |
50 |
50 |
|
Gas Temp (°C) |
40 |
40 |
40 |
|
Plate Temp (°C) |
60 |
60 |
60 |
|
Plate Height |
55 |
55 |
75 |
Figure 3. Evaporation time for semi- or low volatile solvents under static- and gradient plate height.
While high temperatures are crucial for reducing evaporation time, the actual time savings are more pronounced for the low-volatile solvents compared to volatile ones (Figure 4). Most volatile solvents can be evaporated in about 15 minutes, regardless of whether moderate or high-temperature settings are used (Figure 4A). Therefore, we recommend using low to moderate-temperature settings to ensure the thermal stability of analytes/samples.
In contrast, when working with mid to low-volatile solvents, especially those containing water, high temperatures can significantly reduce the evaporation time (Figure 4B). This efficiency improvement is valuable for expediting experiments. However, despite the benefits of increased efficiency, high temperatures may lead to analyte/sample degradation. Therefore, it is important to evaluate the stability of your analytes (or samples) before opting to increase the temperature during evaporation.
When high temperatures must be avoided, increasing the gas flow is an alternative strategy to reduce the evaporation time. Figure 2 illustrates the impact of low-, mid-, and high nitrogen flows (25, 50, 90, L/min) on the evaporation time across different solvents. The efficiency improvement is more notice- able with less-volatile solvents, especially mixtures containing water.
However, strong gas flow can cause liquid splashes and cross- contamination, particularly at the initial stages of evaporation. Therefore, it is advisable to start with low- or mid-gas flow and gradually ramp to the high flow as the liquid volume decreases. Additionally, the cost of nitrogen should be considered in any effective evaporation method. This is especially important for laboratories that rely on nitrogen tanks (or generators), as managing nitrogen consumption can significantly impact overall operational costs.
|
Conditions |
Moderate Temperature setting |
High Temperature setting |
||
|
Step |
1 |
2 |
1 |
2 |
|
Gas Flow (L/Min) |
50 |
50 |
50 |
50 |
|
Gas Temp (°C) |
40 |
40 |
60 |
60 |
|
Plate Temp (°C) |
60 |
60 |
80 |
80 |
|
Plate Height (mm) |
55 |
75 |
55 |
75 |
Figure 4. Evaporation time for (A) volatile solvents and (B) semi- or low volatile solvents under moderate and high temperature settings.
One way to enhance evaporation efficiency is to use the gradient plate height, which allows the system to automatically maintain the optimal distance between liquid and gas. Figure 3 compares the evaporation rates of various solvents under static and gradient plate height evaporation. While the gradient plate height does reduce the evaporation time, its impact is not as significant as that of temperature and gas flow rate. Nonetheless, maintaining a close distance between the gas and liquid surface is suggested to ensure efficient nitrogen consumption.
Analyte crosstalk refers to the cross-contamination that often occurs when using the high-throughput and low-volume 96-well pate format in analytical workflow. A major concern arises during evaporation, where analytes from one well may contami- nate neighboring wells as they are carried along with the volatilized solvent. When the concentration of the analyte in the “hotspot” well is considerably higher than in the surrounding wells, analyte crosstalk can lead to the detection of extraneous signals in adjacent and/or surrounding wells, potentially resulting in cross contamination.
One of the simplest ways to minimize evaporation crosstalk is to avoid overfilling the wells. Filling them to 75% or less of their maximum volume can effectively reduce liquid splash and capillary action, especially for liquids with low surface tension and high analyte sample concentration.
Additionally, starting the evaporation with low gas flow and maintaining an appropriate gas-liquid distance (1-3 cm) is crucial to reduce cross-contamination.
Volatile analytes, such as amphetamine, can co-evaporate with non-polar solvents and migrate to other wells during evaporation. By adjusting the pH of the sample, these analytes can form less volatile and more polar salts, effectively reducing crosstalk. The downside of this approach is that strong acidic or basic conditions may cause hydrolysis or analyte degradation during evaporation. While valuable, this strategy may not work for all volatile analytes, especially those whose volatility is less impacted by pH.
Figure 5. Biotage® ACT Plate Adapter
The Biotage® ACT Plate Adapter (Figure 5) is a novel solution to hot spot cross-contamination during evaporation. This adaptor fits securely on top of the 96-well collection plate with ‘chimneys’ directing the gas flow into the wells during evapora- tion. As the solvent evaporates, the vapors are directed away from sample wells, minimizing eddy formation and reducing the open surface area at the top of the wells. This design helps prevent vapor that carries the analytes from mixing with gas and re-entering adjacent wells, thereby reducing the risk of cross-contamination. The ACT plate adapter's universal design and shape make it a cost-effective accessory that can significantly decrease the likelihood of falsely elevated results in any 96-well-based assay. Made of anodized aluminum, it is easily cleaned between runs to prevent any potential carryover.
When evaporating samples that contain concentrated volatile analytes in a 96-well plate, the Biotage® ACT plate adapter is the preferred choice to prevent crosstalk contamination during evaporation. This approach helps avoid the introduction of strong acids or bases into the sample, thereby reducing analyte degradation. Comprehensive information and data comparisons on these three approaches for crosstalk prevention are provided in the supplementary data.
In the LC-MS/GC-MS sample preparation workflow, scientists often need to dry down sample/analyte eluates and then reconstitute (or derivatize) them for the subsequent analysis. The eluate fractions typically contain one or multiple types of solvents, ranging from nonpolar to polar. For example:
These diverse solvent compositions necessitate different evaporation methods to achieve optimal efficiency and performance.
When developing evaporation methods, it is important to consider the synergetic effects of temperature, gas flow, and stage height. Additionally, the heat sensitivity of the analyte/ sample, the nature of solvents, and the availability of nitrogen supply are key factors in determining the optimal parameters.
By carefully adjusting these parameters, you can optimize the evaporation process for different types of analytes and solvents, ensuring reliable results.
Table 1 summarizes recommended evaporation conditions and approximate evaporation time for the most commonly used solvent system in the bioanalysis workflow. The methods provided and data can be used as starting conditions or references for method development.
Table 1. Recommendation of evaporation program and estimated time for commonly used solvents by the TurboVap® 96 dual system
|
Solvent |
DCM |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
3 |
5 |
3 |
2 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
8 |
5 |
||
|
Solvent |
MeOH |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
6 |
9 |
3 |
8 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
15 |
11 |
||
|
Solvent |
ACN |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
6 |
9 |
3 |
7 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
15 |
10 |
||
|
Solvent |
IPA |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
6 |
12 |
3 |
8 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
18 |
11 |
||
|
Solvent |
EtOAC |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
4 |
4 |
2 |
3 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
8 |
5 |
||
|
Solvent |
Hexanes |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
2 |
3 |
1 |
2 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
5 |
3 |
||
|
Solvent |
DCM/MeOH/NH4OH (80:20:2, v/v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
4 |
6 |
2 |
5 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
10 |
7 |
||
|
Solvent |
DCM/IPA/NH4OH (80:20:2, v/v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
4 |
5 |
2 |
5 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
9 |
7 |
||
|
Solvent |
MTBE |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
2 |
2 |
1 |
1 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
4 |
2 |
||
|
Solvent |
THF |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
2 |
4 |
1 |
4 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
6 |
5 |
||
|
Solvent |
Acetone |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
4 |
5 |
3 |
3 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
9 |
6 |
||
|
Solvent |
5% Formic acid in MeOH |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
13 |
13 |
10 |
9 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
26 |
19 |
||
|
Solvent |
Diethyl Ether |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
2 |
2 |
1 |
2 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
4 |
3 |
||
|
Solvent |
Toluene |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
9 |
9 |
7 |
6 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
18 |
13 |
||
|
Solvent |
DCM/IPA (90:10, v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
6 |
8 |
4 |
5 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
14 |
9 |
||
|
Solvent |
Pyridine |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
14 |
16 |
9 |
11 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
30 |
20 |
||
|
Solvent |
Piperidine |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
14 |
16 |
9 |
11 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
30 |
20 |
||
|
Solvent |
Pentane |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
2 |
3 |
1 |
2 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
5 |
3 |
||
|
Solvent |
HFIP |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
1 |
2 |
|
Gradient time (min) |
4 |
3 |
4 |
2 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
60 |
60 |
|
Plate Temp (◦C) |
60 |
60 |
80 |
80 |
|
Plate height (mm) |
55 |
75 |
55 |
75 |
|
Cost of time (min) |
7 |
6 |
||
|
Solvent |
|
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
3 |
4 |
|
Gradient time (min) |
10 |
20 |
25 |
25 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
40 |
40 |
|
Plate Temp (◦C) |
60 |
60 |
60 |
60 |
|
Plate height (mm) |
55 |
65 |
70 |
75 |
|
Cost of time (min) |
80 |
40 |
||
|
Solvent |
Water |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
3 |
4 |
|
Gradient time (min) |
15 |
25 |
25 |
30 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
40 |
40 |
|
Plate Temp (◦C) |
60 |
60 |
60 |
60 |
|
Plate height (mm) |
55 |
65 |
70 |
75 |
|
Cost of time (min) |
95 |
70 |
||
|
Solvent |
MeOH/Water (50:50, v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
3 |
4 |
|
Gradient time (min) |
10 |
10 |
20 |
17 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
40 |
40 |
|
Plate Temp (◦C) |
60 |
60 |
60 |
60 |
|
Plate height (mm) |
55 |
65 |
70 |
75 |
|
Cost of time (min) |
57 |
50 |
||
|
Solvent |
ACN/Water (50:50, v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
3 |
4 |
|
Gradient time (min) |
10 |
10 |
15 |
20 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
40 |
40 |
|
Plate Temp (◦C) |
60 |
60 |
60 |
60 |
|
Plate height (mm) |
55 |
65 |
70 |
75 |
|
Cost of time (min) |
55 |
45 |
||
|
Solvent |
ACN/Water (30:70, v/v) |
|||
|
Condition A (Low to moderate temperature) |
Condition B (Moderate to high temperature) |
|||
|
Steps |
1 |
2 |
3 |
4 |
|
Gradient time (min) |
20 |
20 |
25 |
25 |
|
Gas flow (L/min) |
50 |
50 |
50 |
50 |
|
Gas Temp (◦C) |
40 |
40 |
40 |
40 |
|
Plate Temp (◦C) |
60 |
60 |
60 |
60 |
|
Plate height (mm) |
55 |
65 |
70 |
75 |
|
Cost of time (min) |
90 |
55 |
||
The amphetamine solution (500µL) at 10,000 ng/ml was aliquoted into positions E5 and D9 (Figure S1) in a 2ml square 96-well plate as hotspots. The same volumes of the DCM/IPA (95:5, v/v) or methanol (without amphetamine) were aliquoted into the rest of the wells in the corresponding plates.
Plates with the same loading map were evaporated with 3 different approaches: a) without crosstalk prevention procedures, b) mixing with 50µL of 0.5% HCl, and c) using the Biotage® ACT plate adapter. All plates were dried under identical conditions: gas flow 50L/min, gas temperature 40 °C, heating temperature 60 °C, and plate height at 40mm. The plates were reconstituted with 500 µL methanol and analyzed on LC-MS/MS using a Shimadzu Nexera UPLC and a Sciex 5500 triple quadrupole mass spectrometer. Samples detected with 10ng/ml or higher amphetamine were deemed to be impacted by crosstalk.
As shown in Figure S1, severe amphetamine crosstalk was observed in neighboring wells when the collection plate was directly evaporated without prevention procedures. The crosstalk effects were worse in the high-volatile solvent (DCM-IPA, 95:5, v/v) compared to the low-volatile solvent (methanol). There were 13 neighboring wells near the hotspot in the DCM-IPA plate being detected with highly concentrated amphetamine (>100 ng/ml, Figure S1A). A typical amphetamine-positive cut-off in urine sample is at 500 ng/mL or above1, with the detection limits of the analytical method at 10-20 ng/ml2. The detected peak amphetamine concentration can range from 5739 to 19172 ng/ml1. If proper prevention procedures are not followed during evaporation, the hotspot crosstalk effect may lead to false-positive identifications. This could negatively impact diagnostic/therapeutic outcomes and even lead to legal disputes. Acidifying samples effectively reduced the crosstalk (Figure S1B). However, this approach does not completely eliminate the crosstalk, as 3-5 wells adjacent to the hotspot still showed >100 ng/ml of the analytes (Figure S1B).
In comparison, the Biotage® ACT plate adapter offered a much better solution for addressing the issue of hotspot crosstalk. The number of wells affected by crosstalk has significantly reduced from 13 to just 2 (Figure S1). More importantly, the detected concen- trations were below 30 ng/ml, a level that is unlikely to impact clinical reporting outcomes. Additionally, the Biotage® ACT plate adapter eliminates the need to add acids or bases to the sample, which simplifies the sample preparation procedures and reduces analyte degradation.
Literature Number: PPS759