Science with Passion
Application No.: VTN0024 Version 1 11/2022
Advantages in online SPE using the 8-port 2-position and multi-injection valve for PAH analysis
Juliane Kramer; kramer@knauer.net
KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38, 14163 Berlin
Rendering: KNAUER
Summary
Solid phase extraction (SPE) is a useful technique to enhance detection limits to analyse even very small amounts of compounds. Most often SPE is carried out manually but coupled to an HPLC system, the so called online SPE technique allows an automated sample analysis. The used system configuration makes it possible to switch between direct injection via autosampler and injection with an auxiliary pump (aux/feed pump) without changing/replumbing the system.
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants and are widespread across the globe mainly due to long-term anthropogenic sources of pollution [1]. Due to their high mutagenic and carcinogenic potential a sensitive determination is mandatory. SPE as a part of sample preparation is often necessary because of low quantification and detection limits that need to be achieved. Most often SPE is carried out manually but when coupled to an HPLC system, the so called online SPE technique allows an automated sample analysis and is a useful technique to enhance detection limits. Another advantage of SPE sample preparation in general lies in matrix reduction which is also beneficial for the limits of detection and quantification.
Fig. 1 Exemplary structures of selected PAHs
With the different switching possibilities of the configured valves, it is possible to manually load a sample, to use an autosampler or to use the auxiliary pump for a larger sample volume without replumbing the system.
Sample Preparation
EPA 610 PAH mixed standard (Sigma Aldrich, product number: 4S8743) came as a ready to use solution. The mixed standard was diluted with acetonitrile:water 60:40 (v/v) to different concentrations.
Results
The method for determination of PAHs described in application note VEV0088 uses a configuration with three different valves. On basis of this application and hardware setup the different switching possibilities will be explained.
Fig. 2 Exemplary chromatogram of 16 PAH according EPA method 610 [2], red – fluorescence trace, blue – UV trace 230 nm, (1) naphthalene, (2) acenaphthylene, (3) acenaphthene, (4) fluorene, (5) phenanthrene, (6) anthracene, (7) fluoranthene, (8) pyrene, (9) benzo[a]anthracene, (10) chrysene, (11) benzo[b]fluoranthene, (12) benzo[k]fluoranthene, (13) benzo[a]pyrene, (14) dibenzo[a,h]anthracene, (15) benzo[g,h,i]perylene, (16) indeno[1,2,3-cd]pyrene
Fig. 3 Used valves in system configuration
With the valve settings displayed in Fig. 4, the main column is operated with the binary gradient pump and an injection via autosampler is possible. Theoretically, the SPE cartridge can be loaded manually from the syringe position of the multi-injection valve. Due to the used particle size of the SPE column and the resulting backpressure, loading the SPE with a syringe can be difficult to implement.
Fig. 4 Manual SPE load; multi-injection valve: Pos 1, 8-port 2-position: Pos 1
When switching the 8-port 2-postion valve to position 2 (Fig. 5) the SPE is now in the flow path of the binary pump and the manually loaded sample will be eluted onto the main column for analysis. When using position 1 of the multi-injection valve, no automated cleaning of the SPE cartridge is possible because it is not connected to the aux pump.
Fig. 5 SPE elution; multi-injection valve: Pos 1, 8-port 2-position: Pos 2
Direct injection using the autosampler is also possible with the following setting: Multi-injection valve: 2, 8-port 2-position: 1. The SPE cartridge cannot be loaded with these conditions. Also, the aux pump would be directly connected to the waste and has no functionalities. With the setting: multi-injection valve: 2, 8-port 2-position: 2, the SPE column is switched into the flow path but because it couldn’t be loaded before, these settings were not usable for the application and are therefore not displayed.
In Fig. 6, the sample is injected onto the SPE column via the autosampler. Dependent on the used syringe and sample loop, different volumes can be loaded. Switching the 8-port 2-position valve to position 2, will result in a connection of the binary pump to waste (W2).
Fig. 6 SPE load via autosampler; multi-injection valve: Pos 3, 8-port 2-position: Pos 1
The settings shown in Fig. 7 were used as starting conditions for the PAH application. The binary pump is connected to the main column. The aux pump is connected to the SPE column and can perform automated conditioning, sample loading and cleaning with the solutions from the multiposition valve.
Fig. 7 SPE load via aux pump; multi-injection valve: Pos 4, 8-port 2-position: Pos 1
Switching the 8-port 2-position valve to position 2 (Fig. 8), the SPE is in the flow path of the binary pump, and the loaded sample will be eluted onto the main column for analysis.
Fig. 8 SPE elution; multi-injection valve: Pos 4, 8-port 2-position: Pos 2
The following tables display the switching positions and aux pump flow rates that were used for the method in application note VEV0088 for loading the SPE via the autosampler.
Tab. 1 Valve switching program, SPE loading/elution/cleaning
Time (min) | Multi-injection valve | 8-port 2-position valve | Multiposition valve 8port, 1/8" | Comment |
0.00 | 4 | 1 | 1* | – |
0.02 | 3 | 1 | 1 | SPE load |
0.20 | 4 | 1 | 1 | SPE wash |
1.00 | 4 | 2 | 1 | SPE elution |
30.00 | 4 | 1 | 1 | SPE cleaning and conditioning |
39.00 | 4 | 1 | 2** | |
42.00 | 4 | 1 | 1 |
* H2O, ** acetonitrile
Tab. 2 Pump program for ASM 2.2L (aux pump), SPE washing/cleaning/conditioning
Time | Flow rate (ml/min) |
0.00 | 0.00 |
0.20 | 0.00 |
0.22 | 3.00 |
1.00 | 3.00 |
1.02–39.00 | 0.00 |
39.02–44.00 | 3.00 |
45.00 | 0.00 |
Conclusion
The multi-injection valve enables three different SPE loading options: manually with syringe, via autosampler, and via feed pump. That makes the method very versatile and allows the user to inject a broad variety of different sample volumes from µl to ml scale. Referring to application note VEV0088, it was also demonstrated that loading higher sample volumes on the SPE cartridge will lead to an enhancement of the method sensitivity. The developed application shows that using special types of valves is both beneficial in terms of automation, time savings in sample preparation and detection limits.
Material and Methods
System configuration
Component | Description | Article No. |
Pump | AZURA P 6.1L HPG, | |
Autosampler | AZURA AS 6.1L, Standard | |
Detector UV | AZURA DAD 2.1L | |
Flow cell | High Sensitivity KNAUER LightGuide UV Flow Cell Cartridge | |
Detector FLD | Shimadzu RF-20A | |
Thermostat | AZURA CT 2.1 | |
Valve | AZURA Valve Unifier VU 4.1 | |
Valve | Multiposition Valve, 8 port, 1/8" | |
Assistant | AZURA Assistant ASM 2.2L | |
Left module: Pump P 4.1S, 10 ml, stainless steel | ||
Middle module: Valve drive VU 4.1 | ||
Right module: Valve drive VU 4.1 | ||
Valve | High-pressure injection Valve, 8 Port 2-position | |
Valve | Multi-injection Valve, DLC stainless steel | |
Column | NUCLEOSIL 100-5 C18 PAH, 5 µm, 150×4 mm | –––––––– |
SPE column | Eurosil Bioselect 300-5 C8. 30×4 mm | 03DK081EBJ |
Software | ClarityChrom 8.5 – Workstation, autosampler control included | |
Software | ClarityChrom 8.5 – PDA extension |
References
[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674206/, 2022/03/21
[2] https://www.epa.gov/sites/production/files/2015-10/documents/method_610_1984.pdf, 2022/03/21
Application details
|
Method |
HPLC |
|
Mode |
RP |
|
Substances |
EPA 610 PAH mixture, fluoranthene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, benzo[g,h,i]perylene, indeno[1,2,3-cd]pyrene |
|
CAS number |
206-44-0, 205-99-2, 207-08-9, 50-32-8, 191-24-2, 193-39-5 |
|
Version |
Application No.: VTN0024 | Version 1 11/2022 | ©KNAUER Wissenschaftliche Geräte GmbH |