Automated fractionation of EPH in soil or water using ISOLUTE® EPH on Biotage® Extrahera™

Written by Biotage | Dec 6, 2025 10:50:27 PM

Introduction

ISOLUTE® EPH SPE cartridges and associated methodologies have been optimized to efficiently fractionate EPHs into aliphatic and polycyclic aromatic (PAH) fractions (C8–C40 aliphatics, C10–C22 aromatics).

The principle is similar to the approach taken by the Massachusetts Department of Environmental Protection (MADEP) and TPH criteria working group (TPHCWG) methods. However, compared to these methods, the ISOLUTE® EPH fractionation cartridge has been significantly reduced in size and the sorbent has been optimized. This allows an automation- compatible method of fractionation which overcomes the common problem of PAH breakthrough into the aliphatic fraction, in addition to reduced solvent volumes.
This application note describes the operating conditions for the automated fractionation of EPH into aliphatic and PAH fractions using the ISOLUTE® EPH cartridge in conjunction with the Biotage® Extrahera™ automation system.

Application analytes

octane	eicosane	dotriacontane
decane	docosane	tetratriacontane
dodecane	tetracosane	hexatriacontane
tetradecane	hexacosane	octatriacontane
hexadecane	octacosane	tetracontane
octadecane	triacontane

naphthalene	fluoranthene	benzo(a)pyrene
acenaphthalene	pyrene	indeno(123,c,d)pyrene
acenaphthene	benz(a)anthracene	dibenz(ah)anthracene
fluorene	chrysene	benzo(ghi)perylene
phenanthrene	benzo(b)fluoranthene
anthracene	benzo(k)fluoranthene

Method overview

Soil and water extraction procedures

Any of the MADEP or TPHCWG approved soil or water extraction procedures can be used with the ISOLUTE EPH method.

The only requirements for successful fractionation are that the final solvent containing the extract should be either pentane or hexane. The presence of any polar modifier in the final extract will compromise the fractionation process. For example, if the soil or water sample is extracted using dichloromethane, this solvent must be exchanged for hexane or pentane prior to fractionation. Up to 1 mL soil or water extract in hexane or pentane can be loaded onto the ISOLUTE EPH cartridges.

Sample preparation procedure

Format

ISOLUTE® EPH 1.45g/3 mL, part number 928-0145-B.

Sample

Soil or water extract in hexane or pentane.

Cartridge conditioning

6 mL Hexane (4 x 1.5 mL).

Sample loading

1 mL Hexane or Pentane extract. Collect cartridge eluate.

Aliphatic elution

1.5 mL Hexane. Add to cartridge eluate from load step.

Aromatic elution

4.5 mL DCM (3 x 1.5 mL). Collect in a separate tube to the aliphatic elution.

Post elution

Gently vortex and homogenize both fractions for each sample and transfer 1 mL of each fraction to separate GC vials for analysis

GC conditions

Instrument

Agilent 7890A with QuickSwap

Column

Agilent J&W DB-5, 30 m x 0.25 mm ID x 0.25 μm

Carrier

Helium 1.2 mL/min (constant flow)

Inlet

300 °C, Splitless, purge flow: 50 mL/min at 1.0 min

Injection

2 µL

Wash solvents

Methanol and DCM

Oven temperature

Described in Table below:

Aliphatics	Aromatics
Initial temperature 45 °C, hold for 1 minute	Initial temperature 45 °C, hold for 1 minute
Ramp 35 °C to 115 °C	Ramp 10 °C to 350 °C
Ramp 70 °C to 350 °C, hold for 6.5 minutes

Post run

Backflush for 2.4 minutes (3 void volumes)

Transfer line

300 °C

Mass spectrometry conditions

Instrument

Agilent 5975C

Source

230 °C

Quadrupole

150 °C

MSD mode

SIM

SIM parameters

Ions were acquired in the Selected Ion Monitoring (SIM) mode.
Aliphatic ions acquired: 41, 43, 57, 71, 85
Aromatic ions acquired displayed in Table below:

SIM Group	Analyte	Target (Quant) Ion	Target (Quant) Ion 1	Target (Quant) Ion 2
1	Naphthalene	128	102	126
2	Acenaphthalene	152	76	150
3	Acenaphthene	153	76	154
4	Fluorene	166	163	165
5	Phenanthrene	178	152	176
5	Anthracene	178	152	176
6	Fluoranthene	202	101	200
6	Pyrene	202	101	200
7	Benz(a)anthracene	228	113	226
7	Chrysene	228	113	226
8	Benzo(b)fluoranthene	252	126	250
8	Benzo(k)fluoranthene	252	126	250
8	Benzo(a)pyrene	252	126	250
9	Indeno(123,c,d)pyrene	276	137	138
9	Dibenz(a,h)anthracene	278	139
9	Benzo(ghi)perylene	276	137	138

Results

The optimized ISOLUTE® EPH protocol on Biotage® Extrahera^TM provided typical recoveries of EPH in soil as demonstrated in Figures 1 and 2, with RSD values <10% for all analytes.

Figure 1. Chart demonstrating typical recoveries of aliphatic analytes in the hexane fraction

Figure 2. Chart demonstrating typical recoveries of aromatic analytes in the dichloromethane fraction

Figure 3. Total Ion Chromatogram of aliphatic analytes in the hexane fraction.

Figure 4. Total Ion Chromatogram of aromatic analytes in the dichloromethane fraction.

Reduction in solvent consumption

ISOLUTE® EPH cartridges allow a significant reduction in solvent consumption compared to the existing MADEP or TPHCWG procedures as is illustrated in the following Table:

Method	Hexane/Pentane (mL)	Dichloromethane (mL)
ISOLUTE EPH	7.5 mL	4.5 mL
MADEP	50 mL	20 mL
TPHCWG	32 mL	30 mL

Additional information

» All solvents were HPLC grade.
» For fully automated workflow, an additional 12x75mm x 24 position collection rack (part 414511SP) is required to collect separate fractions.

Ordering information

Part Number	Description	Quantity
928-0145-B	ISOLUTE® EPH 1.45 g/3 mL	50
For Automated Processing
414001	Biotage® Extrahera	1
414008	Configuration Kit 24 positions	1
414511SP	Collection Rack 12 x 75 mm, 24 positions	1

Appendix

Biotage® Extrahera™ settings

The method described in this application note was automated on the Biotage® Extrahera™. This appendix contains the software settings required to configure Extrahera to run this method. When two collection racks are present, sample processing and fractionation can be fully automated and 24 samples can be processed in a total of 42 min 30 secs.

Method Name: Hydrocarbon Fractionation by ISOLUTE® EPH 1.45 g/3 mL
Sample Plate/Rack: 12 x 75 mm Test Tubes EPH
Extraction Media: ISOLUTE® EPH 1.45 g/3 mL

Literature number: AN877

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