Separation of Homologous Series ofTechnical Detergents with the Agilent
1290 Infinity 2D-LC Solution Coupled
to an Evaporative Light Scattering
Detector
Application Note
Food Testing & Agriculture – Consumer ProductsAuthors

Abstract

Victoria Elsner

This Application Note shows the separation of a series of homologous non-ionic

Institute for Pure and Applied Masssurfactants in a technical product with the Agilent 1290 Infinity 2D-LC Solution

Spectrometry,by combination of a HILIC column in the first dimension and a reversed phase

University of Wuppertal,

column in the second dimension. Surfactants typically do not show activity in UV

Wuppertal, Germany

detection, hence, the Agilent 1260 Infinity ELSD was used. The visualization of theOliver J. Schmitz
Applied Analytical Chemistry,
University of Duisburg-Essen,
Essen, Germany
Volker Wulf
BASF Personal Care and Nutrition
GmbHDüsseldorf, Germany
Edgar Naegele
Agilent Technologies R&D andMarketing GmbH & Co.
KG, Waldbronn, Germany

Verified for Agilent
1290 Infinity II LC

compounds detected by the ELSD is demonstrated with dedicated 2D-LC software.

Introduction
Fatty alcohol ethoxylates belong to theclass of non-ionic surfactants and areused for a large variety of consumer
products like cosmetics, cleaners, colors,
plant protection, textiles and otherindustrial applications. The production
process starts with a homologous seriesof fatty alcohol compounds typically fromlauryl alcohol (C12) to stearyl alcohol(C18). These compounds are reacted with
ethylene oxide, which gives a second
homologous series of ethoxylates at theother side of the molecule (Figure 1).
To monitor the quality and composition of
the technical product, it is important tohave a separation method in hand which
is able to separate both homologous
series, the fatty alcohol component
and the successive series of ethoxylate
units. The only separation technology
which can fulfill this requirement iscomprehensive 2-dimensional liquid
chromatography by combination of
the selectivity of a HILIC and an RP
separation. The other challenge is to findthe right detection method because UV
detection is not possible due to the lackof UV activities of these compounds.
Typically an Evaporative Light Scattering
Detector (ELSD) is used for such aclass of non-UV active and nonvolatilecompounds. In addition, this is acost‑effective detection method useful fortechnical quality control.

C12–C18O
n = degree of ethoxylation (EO)

OHnFigure 1. Structure formula of fatty alcohol ethoxylat compounds.

Experimental
Method
1st Dimension pump
Solvent A

Water, 50 mM ammonium acetate

Solvent B

Acetonitrile

Flow rate

0.025 mL/min

Gradient

0 min - 97 % B, 10 min - 97 % B, 60 min - 85 % B, 100 min - 85 % B,
120 min - 70 % B, 140 min - 70 % B, 160 min - 97 % B

Stop time

160 minutes

Post time
2nd dimension pump

20 minutes

Solvent A

Water, 10 mM ammonium acetate

Solvent BMethanol

Flow rate

3 mL/minInitial gradient

0 min - 50 % B, 0.1 min - 70 % B, 0.65 min - 95 % B, 0.75 min - 95 % B,
0.80 min – 50 % B, 1.00 min – 50 % B

Stop time

1.00 minute

Modulation time
TCC

1.00 minute1st dimension column on the left side at 25 °C
2nd dimension column on the right side at 50 °C
Two 40-µL loops are connected to the 2-Position/4-Port Duo valve and are located on the left side.
Autosampler
Injection volume

5 µL

Sample temperature

8 °C

Needle wash
ELSD

6 seconds in methanol

Evaporator temperature

80 °C

Nebulizer temperature

70 °C

Data rate

40 Hz

Gas flow

1.3 SLM2Equipment

The Agilent 1290 Infinity 2D-LC Solution
consists of:••Agilent 1290 Infinity Binary Pump
(G4220A)
Agilent 1260 Infinity Quaternary
Pump (G-1311B)

Column 1st dimension

SeQuant, Sweden, ZIC-HILIC,
250 × 2.1 mm, 5 µm

Column 2nd dimension

Dr. Maisch GmbH, Germany, Reprospher
C8-Aqua, 30 × 4.6 mm, 5 µm

Chemicals•Agilent 1290 Infinity Autosampler(G4226A) with Thermostat(G1330A)•Agilent 1290 Infinity Thermostatted
Column Compartment (TCC)(G1316C) with 2-Position/4-PortDuo valve (G4236A) for 2D-LC

All solvents used were LC grade.
Acetonitrile and methanol werepurchased from Merck, Germany. Freshultrapure water was obtained from a
Milli-Q Integral system equipped with a
0.22-μm membrane point-of-use cartridge(Millipak). All chemicals used as standard
were purchased from Sigma-Aldrich,Germany.

•Agilent 1260 Infinity ELSDSample

SoftwareOpen Lab CDS ChemStation Rev. C01.03
with 2D-LC add-on software. LCxLCSoftware for 2D-LC data analysis from GC
Image LLC., Lincoln, NE, USA.

A synthetic, technical, fatty alcohol
ethoxlyate with an alkyl chain from C12
to C18 was dissolved in acetonitrile/water 80/20 (v/v) and diluted to the final
concentration of 3 mg/mL.

Results and Discussion
The comprehensive 2-dimensional
separation of the technical mixture offatty alcohol ethoxylates was done on aHILIC phase in the first dimension undertypical HILIC conditions. The seconddimension separation was done on areversed phase column under typical RP
conditions. The HILIC phase in the first
dimension separates the compounds by
their increasing degree of ethoxylation
for compounds with 3 to 20 ethoxy
units. The reversed phase column in thesecond dimension separates the fractionof ethoxylate with a defined degree ofethoxylation by their fatty alcohol chain
length by increasing number of carbon
atoms (Figure 2).
The 2-dimensional separation spaceis nearly perfectly orthogonal. It starts
with the lowest carbon chain length andlowest degree of ethoxylation at thelowest retention time in first and seconddimension time. The second dimension
retention time increases by chain lengthand the first dimension retention timeincreases by ethoxylation yielding thehighest 2-dimension retention time forlong chain compounds with highest
degree of ethoxylation (Figure 2).

Figure 2. 2-dimensional separation of a technical mixture of fatty alcohol ethoxylates of fatty alcohols C12 to C18 and a degree ofethoxylation 3 to 20 (see formula in Figure 1).

3

The analysis under given conditions
shows perfect separation for eachcompound in the mixture and allows
its characterization. The ELSD is able
to acquire data at a data rate sufficient
to follow the fast chromatographic
separation in the second dimension.
For a product mixture which was wellcharacterized in advance, the main
compounds can be confirmed easily for a
production batch. Lower level compounds
can be seen on a lower signal intensity
level. For a more reliable confirmation, amore sensitive detection such as mass
spectrometry is required (data not shownhere).

Conclusion
This Application Note demonstrates
the capability to run the Agilent 1290
Infinity 2D-LC Solution with an ELSD asa detector for non-UV active compounds.
This can be used for the separationand detection of complex oil and fatty
alcohols or acids and their derivatives.
These compounds are important technical
basic chemicals typically occurring incomplex mixtures from the production
process and, hence, 2D-LC separation
could be used for quality control ofproduction batches.

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This information is subject to change without notice.
© Agilent Technologies, Inc., 2013-2014
Published in the USA, September 15, 2014
5991-2775EN