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

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).

 

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.

 

 

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