Solid Phase Extraction (SPE) is a well-established technique used in analytical chemistry for the separation of analytes from complex matrices and offers several significant advantages over other sample preparation techniques, including improved selectivity for target analytes, the ability to pre-concentrate samples, improved accuracy and improved sustainability credentials compared to other techniques.

One of the critical factors in the success of an SPE method is the selection of an appropriate stationary phase (sorbent).  Originally, sorbents such as bare silica and florisil were used alongside silica chemically modified with bonded phases typically seen in HPLC, such as C18.  The choice of stationary phase is based on an initially somewhat arbitrary matching of the chemistry of the SPE sorbent with that of the analyte, to ensure good retention of the target analyte or analyte groups.  Typically LogP or LogD values are used to estimate target analyte hydrophobicity and this is used alongside analyte functional group chemistry to select candidate sorbents for SPE method development.

More recently Hydrophilic-Lipophilic Balance (HLB) SPE sorbents have been developed.  These phases are based on a copolymer of hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene (Figure 1) and offer a unique combination of properties that enhances the performance of SPE.  As HLB phases are polymeric, they can be manufactured as mono-disperse particles, improving the efficiency of extraction and hance the resulting elution volume, which can lead to increased sensitivity of the method.  The ratio of the hydrophilic and hydrophobic moieties is optimized to provide retention for the widest range analytes.

Figure1. HLB Divinylbenzene/N-vinylpyrrolidone copolymer

Figure1. HLB Divinylbenzene/N-vinylpyrrolidone copolymer

Versatility in Analyte Retention

Probably the biggest advantage of HLB is that it can selectively retain a wide range of compounds from polar to non-polar due to its balanced hydrophilic and lipophilic properties.  HLB can retain acids, neutral and basic compounds within the same sample.

The hydrophobic divinylbenzene head group retains hydrophobic targets and the hydrophilic N-vinylpyrrolidone head retains polar targets efficiently (Figure 2).  This makes HLB an excellent choice for extracting a broad range of analytes from diverse sample matrices and is a highly popular sorbent for the extraction of analyte suites in the environmental, biological and food industries.

Bond Elut HLB demonstrates good recovery for a panel of targets from polar to non-polar with MeOH elution.

Figure 2. Bond Elut HLB demonstrates good recovery for a panel of targets from polar to non-polar with MeOH elution.

For some non-polar targets, stronger solvents such as 1:1 MeOH/CAN, CAN and ethyl acetate can be useful to improve recovery

HLB phases also demonstrate superior retention of polar analytes compared to silica-based sorbents and other polymers used in SPE (Figure 3).

Figure 3. Recoveries of Hydroxymetronidazole-a polar compound with logP: -1.3, using a generic method with 100% methanol elution, on Agilent Bond Elut HLB.  

Figure 3. Recoveries of Hydroxymetronidazole-a polar compound with logP: -1.3, using a generic method with 100% methanol elution, on Agilent Bond Elut HLB. Far greater recoveries than a non HLB polymer can be seen.

HLB phases have several key features compared to other SPE media including:

Highly Water Wettable

Due to the hydrophilic component of HLB, the phase is more water wettable than traditional silica-based phases.  Water is easily drawn into and retained by the porous structure and the material is much less susceptible to drying out under vacuum once the protocol solvents have been eluted.  This has a significant practical benefit for the analyst, as samples processed using vacuum manifolds or positive pressure manifolds may be left unattended for short periods.  This allows an analyst to leave the samples unsupervised and perform other laboratory tasks.  It’s a simple benefit but can have a significant effect on the efficiency of the laboratory.

Higher Capacity

HLB demonstrates higher adsorption capacity and loadability compared to C18 bonded silica sorbents.  As a rule of thumb, we would expect a silica-based sorbent to have a capacity of 5% of the bed mass-so a 100mg bed mass cartridge would be capable of retaining 5 mg of analyte approximately.  For HLB phases, we would expect to see a capacity of 10% or greater.  The advantage to the analyst is the ability to use a smaller bed mass cartridge compared to silica for the same extraction.  This presents not only significant cost benefits, but also results in lower analyte elution volumes, improving the sensitivity of the technique.

pH Stability

Silica based sorbents, just like silica based HPLC columns, have a pH stability range of approximately 2-8.  If we work with protocol solvents above pH 8 we risk dissolving the silica, and below pH 2 there is a risk of hydrolysis of the bonded phase.  HLB is stable over a pH range of 1-14, which offers greater flexibility for the analyst in development of methods, allowing manipulation of loading and elution solvent pH to adjust the extent of ionization of the analyte, thus improving the selectivity of the sample preparation protocol.  

Improved Reproducibility

Silica SPE media are often made from irregular shaped particles, whereas the HLB phase is monodisperse.  Spherical particles, just like in HPLC, are more efficient and reproducible than irregular particles.  The HLB SPE process is therefore more predictable and reproducible and again offers the opportunity to elute analytes with smaller solvent volumes.  Additionally, the polymeric HLB contains no free surface silanol moieties, which can introduce variability into the sample preparation method.  With a more reproducible method there is less need for repeat sample analysis and this can have huge benefits in terms of analysis time, cost and solvent savings, improving both laboratory efficiency and sustainability.

Method Simplicity

The basic method for the HLB SPE Sample Prep Workflow is extremely straight forward (Fig 4).  From this starting point the protocol can be further optimised.  Generally, the concentration of methanol in the wash solution will be increased as high as possible, prior to the target analyte(s) being lost in the wash solution, ensuring a clean extract.  If analytes of interest are highly polar, 100% aqueous can be used as a wash solvent.  The organic composition of the elution solvent may be adjusted to a level just high enough to elute all of the target analytes from the sorbent, again ensuring the cleanest extract.  For extremely hydrophobic analytes, acetonitrile can be used in place of methanol as a stronger eluotropic solvent.

If analytes of interest are ionizable, it can be beneficial to adjust the pH of the conditioning and loading solutions to ensure that the analyte is in its neutral form in order to increase recoveries.  Acids will be best retained by loading in solvent adjusted to 2pH units below their pKa, whilst bases will show best recoveries by using a loading solvent adjusted to 2 pH units above their pKa.

Generic HLB Starting Method

Figure 4.  Generic HLB Starting Method

Click here to read an application note describing the analysis of a suite of sulfonamide drugs in water, extracted using Agilent Bond Elut HLB cartridges and analysed by LC/MS/MS.  

The study developed and validated a method for the quantitative analysis of 19 sulfonamide drugs and metabolites, producing excellent calibration linearity and high sensitivity.  The use of Bond Elut HLB for sample preparation produces a highly accurate and precise method.


Hydrophilic-Lipophilic Balance (HLB) SPE sorbents offer several advantages over silica and other polymer based SPE products.  For new SPE method development, HLB should be the first-choice media considered, since it offers excellent recoveries for both polar and non-polar compounds, increased reproducibility and considerable cost and practicality benefits.  For older methods validated on silica-based phases, where recoveries and reproducibility are poor, it would be well worth considering redeveloping the method to take advantage of HLB sorbents.

Contact Element Laboratory Solutions sales representative to discuss your SPE method.  We can support with free Agilent Bond Elut HLB sample cartridges and advice on developing your HLB method.