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Customer Application: Optimization of a Real Pesticide Method for Routine Analysis — Faster and More Sensitive on HPLC, More Robust on UHPLC

29 Mar 2024

When we introduced our Raptor ARC-18 core-shell LC columns in 2014, we were able to immediately solve an urgent problem for a customer. As this example clearly shows how 2.7 µm and 5 µm Raptor core-shell particles can easily improve HPLC and UHPLC methods, we would like to share this information with you as well.

Our customer (SGS INSTITUT FRESENIUS GmbH, Taunusstein, Germany) analysed 130 pesticides (see below) in different waters (mineral and drinking water, well and surface waters, raw and process water, waste water) by direct injection of 50–100 µL of sample on HPLC-MS/MS (up to max. 400 bar) and UHPLC-MS/MS (up to max. 600 bar). Both methods were optimized by switching from fully porous particle (FPP) RP columns to a 2.7 µm Raptor ARC-18 superficially porous particle (SPP, core-shell) column (see table below). The sensitivity of the HPLC method (so far on a 5 µm fully porous column) could be increased and the run time accelerated while maintaining the pressure. The UHPLC method (so far on a 1.7 µm fully porous column) became more robust and was even accelerated, while achieving the same separation performance at lower pressures.

  HPLC-MS/MS with fully porous column HPLC-MS/MS with Raptor ARC-18 Core-Shell Column UHPLC-MS/MS with fully porous column UHPLC/MS with Raptor ARC-18 Core-Shell Column
Column Length [mm] 150 100 100 100
ID [mm] 2.1 2.1 2.1 2.1
Particle Size [μm] 5 2.7 1.7 2.7
Temperature [°C] 25 25 45 30
Mobile Phase A A: 90% water,
10% methanol,
5mM buffer *
A: 90% water,
10% methanol,
5mM buffer *
A: 90% water,
10% methanol,
10mM buffer *
A: 90% water,
10% methanol,
10mM buffer *
Mobile Phase B B: 100% methanol,
5mM buffer *
B: 100% methanol,
5mM buffer *
B: 100% methanol,
10mM buffer *
B: 100% methanol,
10mM buffer *
Gradient ** 0 - 89% B 0 - 89% B 27 - 100% B 15 - 100% B
Flow [mL/min] 0.3 0.3 0.25 0.4
Pressure [bar] 150 - 350 150 - 300 450 - 570 250 - 350
Injection Volume [μL] 50 50 100 100
Run time (until the end of the separation) [min] 11.4 3.3 13.5 7.8
Total Cycle Time (until the next injection) [min] 19.5 10 19.5 10.5
Advantages by using a Raptor ARC-18 Core-Shell Column   Sharper peaks, better sensitivity, shorter analysis time   Same separation efficiency, but longer column life time, lower pressure, shorter analysis time

* Buffer = ammonium formate; ​** Gradient details on request

So far only a 0.2 µm precolumn filter could be used in front of the column for both instrument types. Using a guard column for better protection in case of waste water samples was not possible, because the pressures were already close to the limits of the systems (400 and 600 bar). Whereas the Raptor column could be used with a corresponding guard column due to the lower pressures achieved within the final method. By the way, Raptor 2.7 µm columns can also be used up to 600 bar in continuous operation if needed.

UHPLC with particles < 2 µm can certainly be the ultimate solution, but the success strongly depends on the cleanliness of the injected samples or extracts. The smaller the particle diameter, the smaller the interstitial spaces in the column, the higher the risk of clogging. Working with < 2 µm particles can be a challenge in routine analysis with varying sample types. Often laborious sample preparation is required, or the column has to be replaced frequently.

For this reason, Raptor 2.7 µm core-shell particles are a very good alternative to < 2 µm fully porous particles: the pressure and robustness is comparable to 3 µm columns, but as a result of the special particle design, their separation performance is comparable to < 2 µm fully porous particles.

Particularly important in such multi-methods is the balanced distribution of the analytes over the entire run time in order to achieve optimum sensitivity in MS. If too many compounds elute simultaneously, the sampling time per analyte in MRM mode becomes too short, which reduces sensitivity. The "balanced retention profile" of the Raptor ARC-18 phase fulfills this requirement very well (Figure 1).

Figure 1: LC-MS/MS run of 130 pesticides on a Raptor ARC-18 core-shell column.

LC-MS/MS run of 130 pesticides on a Raptor ARC-18 core-shell column.

 

The robustness and stability of Raptor ARC-18 core-shell columns is shown in the following comparison (Figures 2 and 3): Four compounds were selected as examples, which elute in the early and late eluting areas of the chromatogram. All peaks show excellent retention time stability and consistent symmetrical peak shape even after 5000 injections.

Figure 2: Desisopropylatrazine, Fenuron, AIPA (anthranilic acid isopropylamide), and Diazinon on a new Raptor ARC-18 column.

Desisopropylatrazine, Fenuron, AIPA (anthranilic acid isopropylamide), and Diazinon on a new Raptor ARC-18 column.

 

Figure 3: Desisopropylatrazine, Fenuron, AIPA (anthranilic acid isopropylamide), and Diazinon on Raptor ARC-18 after about 5.000 injections (a wide variety of water samples, from ultrapure water to highly contaminated waste water) — excellent retention time stability and consistent symmetrical peak shape.

Desisopropylatrazine, Fenuron, AIPA (anthranilic acid isopropylamide), and Diazinon on Raptor ARC-18 after about 5.000 injections (a wide variety of water samples, from ultrapure water to highly contaminated waste water) — excellent retention time stability and consistent symmetrical peak shape.

 

List of Components

2,6-Dichlorobenzamide
Acephate
Alachlor
Aldicarb
Aldicarb-sulfone
Aldicarb-sulfoxide
Ametryn
Anthranilic acid
    isopropylamide
Atrazine
Azinphos-ethyl
Azinphos-methyl
Azoxystrobin
Benomyl
Brodifacoum
Bromacil
Buturon
Carbaryl
Carbendazim
Carbetamide
Carbofuran
Chlorobromuron
Chlorfenvinphos
Chloridazon
Chloroxuron
Chlorotoluron
Coumatetralyl
Cyanazine
Cymoxanil
Demeton-S-methyl
Desethylatrazine
Desethylterbuthylazin
Desisopropylatrazine
Desmedipham
Desmetryn
Diazinon
Dichlofluanid
Dichlorvos
Diflubenzuron
Diflufenican
Dimefuron
Dimethachlor
Dimethenamide
Dimethoate
Disulfoton
Diuron
Epoxiconazole
EPTC
Ethidimuron
Ethofumesate
Ethoprophos
Ethylene thiourea
Famphur
Fenamiphos
Fenpropimorph
Fenthion
Fenuron
Flazasulfuron
Flufenacet
Fluometuron
Flusilazole
Flutriafol
Fonofos
Hexazinone
Imidacloprid
Iso-Chloridazon
Isoproturon
Lenacil
Linuron
Malathion
Metalaxyl
Metamitron
Metazachlor
Methabenzthiazuron
Methacrifos
Methidathion
Methiocarb
Methomyl
Methoprotryn
Metobromuron
Metolachlor
Metoxuron
Metribuzin
Mevinphos
Molinate
Monocrotophos
Monolinuron
Monuron
Neburon
Oxamyl
Penconazol
Pencycuron
Phenmedipham
Phosalon
Pirimicarb
Pirimiphos-ethyl
Pirimiphos-methyl
Prometryn
Propachlor
Propazine
Propetamphos
Propiconazole
Propoxur
Propylen thiourea
Prothioconazole
Pyrazophos
Pyrimethanil
Quinalphos
Rimsulfuron
Sebuthyazine
Simazine
Tebuconazole
Terbuthylazine
Terbutryn
Thiacloprid
Thiamethoxam
Tolylfluanid
Triadimefon
Triazophos
Trichlorfon
Trietazin

 

Do you need technical support, a quotation, or would you like to convince yourself of the quality of Restek's LC columns by a trial without any obligation? Contact the European LC Team or, if you are outside of Europe, contact your local Restek representative at www.restek.com/contact-us