C190-E108

Principles and Practical Applications

of Shimadzu's ELSD-LT 2
Evaporative Light Scattering Detector
Technical Repor t vol.6
1. Int roduct ion
In the history of high-performance liquid chromatographs, which dates to the
early 1960s, refractive index detectors (RI detectors) have often been used as
general-purpose detectors. RI detectors enable the detection of components
that do not possess UV absorbance and give a proportional relationship
between the heights of detected peaks and the quantities of detected
components. So, in comparison with absorbance detectors (UV detectors),
they offer advantages such as the ability to ascertain unknown component
quantities and obtain molecular weight distributions for macromolecules. On
the other hand, they also have various disadvantages. For example, they
cannot be used for gradient analysis, the baselines they produce are
susceptible to the influence of fluctuations in the ambient temperature, their
sensitivity is low compared to that of UV detectors, and they are prone to
giving negative peaks, which make quantitative analysis difficult. Furthermore,
with both UV and RI detectors, in cases where the solvent peaks of the
analyzed samples appear at the start of the chromatogram, it is sometimes
not possible to detect target substances with short elution times. Therefore, RI
detectors cannot truly be described as general-purpose detectors. The
principle of evaporative light scattering detectors (ELSD), which solve these
problems, is extremely simple. The target components are converted to a fine
spray by a nebulizer and heated so that only the mobile phase is evaporated.
Light is directed at the remaining target substances and the scattered light is
detected. ELSDs can detect almost all components that are less volatile than
the mobile phase. These detectors first appeared in 1966, but were
subsequently overshadowed by high-performance liquid chromatographs,
which advanced significantly at that time. They were first commercialized in
the early 1980s, and the basic technology of modern-day ELSDs was
established in the mid-1980s. In addition to describing the operating principles
and practical benefits of ELSDs, this article uses the ELSD-LT 2, a product
that achieves greater sensitivity, speed, and convenience than conventional
products by incorporating the latest technology, to present application
examples that utilize ELSD characteristics.

2 . W h a t I s a n E va p o ra tive L ig h t Scat t ering Det ect or ( ELSD) ?

The target substances separated in a column are, together
with the mobile phase, converted to a fine spray by a
nebulizer, and this spray is carried to a drift tube. In the drift
tube, heat is applied so that only the mobile phase is
evaporated. The remaining target substances that were in the
mobile phase are converted to minute solid particles and are
carried to the detection unit. In the detection unit, the target
substance particles cause the light emitted from a light
source to be scattered. This scattered light is measured by a
photomultiplier and the target substances are thereby
detected. The intensity of the signal detected in the ELSD can
be represented by the following equation:
(Signal intensity) = a x (Quantity of target substance)b

1. Separation with HPLC

2. Nebulization

The mobile phase is converted

into a spray of minute droplets
using a gas stream.

Here, "a" and "b" are constants that are determined by a

variety of factors, such as the size of the particles, the
concentration and type of the target substances, the gas flow
rate, the mobile phase flow rate, and the temperature of the
drift tube. In principle, ELSDs are capable of analyzing all
substances that have an evaporation temperature lower than
that of the mobile phase, and can attain roughly the same
level of detection sensitivity for any compound. For this
reason, they are well-suited to the detection of components
such as sugars, fats, surfactants, synthetic macromolecules,
and steroids, as these components have low light
absorbance, making them difficult to detect with UV
detectors.
If the target substances are nonvolatile, detection is possible
down to the nanogram level in nearly all cases.

3. Evaporation of mobile phase

4. Detection

The droplets are carried into a

heated drift tube where the
mobile phase evaporates and
the target components dry and
are converted into minute
particles.

The scattered light created by

the collision of light with the
minute particles that emerge
from the drift tube is detected.

F l ow o f a n a l y s i s
Fig. 1 Flow of ELSD Analysis

One of the most important considerations when performing high-sensitivity analysis

with an ELSD is the set temperature of the drift tube. In particular, when analyzing
semi-volatile substances with low boiling points, the intensity of the signals obtained
varies greatly with this set temperature. If it is too high, semi-volatile substances
may be partially or completely evaporated together with the mobile phase;
consequently, and the quantity of minute solid particles that scatter light may
decrease. This may lead to a reduction in detection sensitivity or make detection
completely impossible. In such cases, decreasing the set temperature of the drift
tube increases the number of target substance particles present after evaporation,
and makes it possible to attain a higher signal intensity. Fig.2 illustrates the
differences in the peak heights that are obtained for caffeine, which is a semivolatile substance, at different set temperatures. It can be seen that the peak height
for a set temperature of 30C is roughly 10 times the peak height for a set
temperature of 50C.
Fig. 2 Variation in Sensitivity for
Different Set Temperatures

3 . E L SD s a n d R I D etec to rs
Like RI detectors, ELSDs are classified as general-purpose
detectors but they differ from RI detectors in the following
ways:
1. They are 5 to 10 times more sensitive than RI
detectors.
2. They support the use of the gradient elution
method.
3. They are not easily affected by changes in the
ambient temperature.
4. They are not affected by interference due to
solvent peaks.
5. Time is not required to allow for the instrument and
the baseline to stabilize.
Although the gradient elution method is effective for the batch
analysis of, for example, multiple components in natural
products, RI detectors cannot be used for this because of
fluctuations in the baseline caused by changes in the
refractive index of the mobile phase.

With ELSDs, baseline fluctuations do not occur in gradient

elution, meaning this method can be used to perform the
efficient, high-sensitivity analysis of multiple components.
This feature of ELSDs is useful for the following types of
analysis:
1. The analysis of compounds that cannot be
detected with UV detectors:
Carbohydrates (sugars), sugar alcohols,
alcohols, terpenoids, surfactants, natural
macromolecules, and synthetic macromolecules
2. The batch analysis of compounds for which the
gradient elution method is difficult to use
because absorbance occurs only in the short
wavelength region:
Fats, phospholipids, glycerides, fatty acids,
natural macromolecules, and synthetic
macromolecules
Also, because ELSDs can be applied to all aspects of the
methods used for LC/MS, including the mobile phases, they
can be used as substitute detectors in place of expensive
LC/MS instruments in the screening of compounds.

Table 1 Analytical Conditions

Previous ELSD

RI detector

Sample

Fructose, Glucose
Sucrose, Maltose

Column

Asahipak NH2P-50 (250mmL. x 4.6mm i.d.)

Mobile Phase

Acetonitrile/ Water = 7/3 (v/v)

Flow Rate

1mL/min

Detection(ELSD)

Temperature: 35C
Gas Pressure: 350kPa

Fig. 3 Variation in Sensitivity for Different Detectors

4 . Fe a t u re s o f E L S D -L T 2
4 -1 . H i g h - Se ns itivity D etec tio n of Semi- Volat ile Subst ances
A c h i e v e d with L o w-T e mp erat ure Evaporat ion of M obile Phase
Nebulizer
Glass cell
Mobile
phase
Nebulizer gas
To the
drift tube

To the drainage outlet

Quantity of droplets

With an ELSD, the larger the nebulized droplets,

the higher the evaporation temperature must be
set in order to evaporate them. If analysis is
performed at a low temperature, the larger
droplets that are not evaporated create scattered
light that gives rise to a high level of noise. The
ELSD-LT2 solves this problem by incorporating a
glass cell with a unique structure. Minute droplets
that leave the ELSD-LT2 nebulizer are carried by
the nebulizer gas stream through the glass cell
and into the drift tube. Larger droplets, however,
are not carried by the nebulizer gas stream and
adhere to the inside surface of the glass tube,
where they change to liquid form. This liquid
accumulates in a siphon tube and is subsequently
discharged. There is always waste liquid in the
siphon section, so all of the nebulizer gas flows
into the drift tube (siphon split method). In this way,
the larger droplets that cause noise are separated
selectively, and the smaller droplets are efficiently
carried into the drift tube.
This technology makes it possible for the ELSDLT2 to suppress noise, even at low evaporation
temperatures. Because mobile phases can be
evaporated at low drift tube set temperatures in
the range of 35C (organic solvent mobile phases)
to 40C (aqueous mobile phases), efficient, highsensitivity analysis is possible for nearly all
compounds.
Also, with the ELSD-LT2, assisting gas is
projected in a cylindrical shape centering on the
drift tube outlet. This increases the concentration
of the target substances that reach the detection
unit from the drift tube and, consequently,
increases sensitivity.
In this way, with both a reduction in noise achieved
through low-temperature evaporation technology
and an increase in peak intensity achieved through
superior detection technology, the ELSD-LT2
realizes high-sensitivity detection.

To the drift tube

To the drainage outlet

Small

Size of droplets

Large

Fig. 4 Structure of Nebulizer and Glass Cell

(Siphon Split Method)

Photomultiplier

Light source

Assisting gas

Assisting gas

Fig. 5 Assisting Gas Focusing (Detection Unit)

The assisting gas that merges with the flow of sample particles just
before they reach the detection unit causes the particles to converge
on the focal point of the light source, en high-sensitivity detection.
Also, because the particles are not dispersed in the detection unit,
there is little contamination of the measurement system, and frequent
maintenance is not required.

4 -2 . M o re U s er F rien d ly
Setting the drift tube temperature and gain is the
only preparation required to perform analysis with
the ELDS-LT2. It is also possible to turn off the
lamp and stop the nebulizer after the completion
of analysis using the auto power-down function.
The use of a long-life LED lamp

and the auto power-down function enable

reductions in the frequency of lamp replacement
and the consumption of nebulizer gas, and thereby
making it possible to reduce running costs.
Furthermore, an automatic cleaning function for
the drift tube helps make maintenance easier.

5 . H i gh -S p ee d A n aly s is Using t he ELSD- LT 2

The ELSD-LT2 supports ultrahigh-speed analysis,
which has received much attention in recent years,
using the technology described below. Backed by the
detecting versatility that is characteristic of ELSDs, it
enables efficient execution of the batch analysis of
multiple components in natural products, something
that calls for ultrahigh-speed analysis.
1. High-speed sampling rate that helps produce
sharp peaks (20 msec)
2. Suppression of peak spreading by reduction of
the drift tube's innert diameter
3. Peak focusing using assisting gas
Furthermore, the siphon split method, which ensures
that only a fine mist enters the drift tube, and the
assisting gas focusing function ensure that
contamination of the detection unit is kept to an
absolute minimum, and that there is no loss of
sensitivity, even in the continuous analysis of multiple
samples. Using the ELSD-LT2 in combination with
the Prominence UFLC series and the Shim-pack XRODS high-speed separation column ensures optimal
performance.

Fig. 6 Ultrahigh-Speed Analysis Based on Four

Semi-Volatile Substances (Alkyl Parabens)
Table 2 Analytical Conditions
Column

Shim-pak XR-ODS (75mmL. x 3mm i.d.)

Mobile Phase

Acetonitrile/ Water =6/4 (v/v)

Flow Rate

1mL/min

Column Temp.

30C

6 . E xa mp les o f A n a ly s is Using t he ELSD- LT 2

Analysis of Polyethylene Glycol
Polyethylene glycol has little UV absorbance and is
therefore difficult to analyze with a UV detector. In
general, an RI detector is used for the analysis of
such compounds. However, when separating lowmolecular-weight polyethylene glycol according to the
polymerization degree using the reverse-phase mode,
the separation achieved with isocratic elution using an
RI detector is limited, and a relatively long time is
required for analysis. Fig. 7 shows an example of the
results obtained by subjecting polyethylene glycol
1000 to gradient elution using the reverse-phase
mode, and performing detection using an ELSD. This
example shows how using an ELSD makes it possible
to separate polyethylene glycol according to the
polymerization degree with a stable baseline using the
gradient elution method.

Fig. 7 Example of Analysis of Polyethylene Glycol

Table 3 Analytical Conditions
Sample

Polyethylene Glycol 1000

Column

Shim-pack VP-ODS (250mmL. x 4.6mm i.d.)

Mobile Phase

A) Water B) Methanol

Time Program

B Conc.40% (0min)

Flow Rate

1.0mL/min

Column Temp.
Detection (ELSD)

60% (15min)

40C
Temperature : 40C
Gas Pressure : 350kPa

Analysis of Triglycerides
Triglycerides, which are the main components of cooking oil,
exist in many different forms that vary according to the acyl
group, and the separation of these components is required
for purposes such as quality control. Although detection is
possible for triglycerides based on the absorbance of the
acyl group in the range of 200 to 210 nm (UV), with systems
that use acetone as the mobile phase, there is a high level of
background absorbance, and UV detectors cannot be used.
Fig. 8 shows an example of the results obtained by
subjecting sesame oil to gradient elution using an acetonebased mobile phase in the reverse-phase mode, and
performing detection using an ELSD. This example shows
how an ELSD can be an effective detection tool even in
cases where the mobile phase has UV absorbance.

Analysis of Oligosaccharides
In the separation of oligosaccharides according to the
glucose polymerization degree, although the normal-phase
mode (HILIC mode) demonstrates superior selectivity, the
time required for elution increases greatly as the number of
glucose units increases. With an RI detector, gradient elution
cannot be used, so analysis must be performed repeatedly
using mobile phase compositions that are suitable for the
separation of each polymerization degree. Fig. 9 shows an
example of the results obtained by subjecting
maltooligosaccharides to gradient elution using the normalphase mode, and performing detection using an ELSD. Using
an ELSD makes it possible to identify maltooligosaccharides
with glucose polymerization degrees of up to around 20.

Fig. 8 Analysis of Sesame Oil

Fig. 9 Analysis of Maltooligosaccharides

Table 4 Analytical Conditions

Table 5 Analytical Conditions

Column

Shim-pack VP-ODS (250mmL. x 4.6mm i.d.)

Sample

Maltooligosaccharides

Mobile Phase

A ) Acetonitrile
B ) Acetone

Column

Asahipak NH2P-50(250mL. x 4.6mm i.d.)

Time Program(40min)

B Conc. 50% (10min)

Mobile Phase

A ) Acetonitrile/Water=7/3 (v/v)
B ) Acetonitrile/Water=4/6 (v/v)

Flow Rate

1.0mL/min

Time Program

B Conc.0% (0min)

Column Temp.

30C

Flow Rate

1.0mL/min

Temperature : 35C
GAIN : 5
Nebulizer Gas : Air
Gas Pressure : 350KPa

Column Temp.

40C

Detection (ELSD)

Detection (ELSD)

Temperature : 40C
Gas Pressure : 350kPa

70% (40min)

100% (25min)

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