For Internal Use Only
PL-ELS 2100 & PL-ELS 2100 Ice
Evaporative Light Scattering Detectors
Introduction
The PL-ELS 2100 and PL-ELS 2100 Ice (Integrated Cooled Evaporator) Evaporative
Light Scattering Detectors are universal detectors for liquid chromatography. They
are designed and built at Polymer Laboratories’ facility in Shropshire, UK, now a part
of Varian, Inc. The unique design combines high sensitivity with low temperature
operation.
Figure 1: PL-ELS 2100/2100 Ice Evaporative Light Scattering Detector
The PL-ELS 2100/2100 Ice is designed for analytical applications where compounds
lack a UV chromophore or fluorophore and are thermally sensitive, particularly in the
pharmaceutical sector. The PL-ELS 2100/2100 Ice is complementary to LC-MS as
both techniques share similar chromatographic requirements.
Principle of Operation
The operation of the PL-ELS 2100/2100 Ice consists of three consecutive steps,
namely Nebulization, Evaporation and Optical Detection (see Figure 2).
During the nebulization stage, the mobile phase is passed through a fine needle and
combined with a gas, such as nitrogen or air, to form a plume of uniform aerosol
droplets. The size and shape of the droplets* determine the sensitivity and
repeatability of the detector. The droplet size distribution is influenced by changes in
the nebulization gas flow as well as other factors, such as mobile phase composition.
Large droplets are removed by condensation or collision with the chamber walls, and
subsequently directed to a waste outlet. The nebulization process can be heated
independently of the evaporation tube, useful in minimizing changes in mobile phase
viscosity during gradient elution.
The aerosol plume formed during nebulization then passes through a heated coil or
tube where the solvent is evaporated, leaving residual particles of the analyte. The
*
Please note, for purposes of clarity, droplet refers to a droplet of mobile phase liquid formed during
nebulization. Particle refers to the solid form of the analyte during the detection process.
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temperature of this heated tube is controlled by the user and is commonly set
according to the mobile phase composition or compound volatility. It is important to
use as low a temperature as possible for this step, to minimize sample volatilization.
Typically, low temperature evaporation is required for low molecular weight
pharmaceuticals. However, this type of compound is analyzed in highly aqueous
eluents, making their detection at ambient temperature difficult with other ELS
detectors. To overcome this problem, the PL-ELS 2100/2100 Ice uses additional dry
gas during the evaporation step to facilitate desolvation without increasing
temperature. This 'evaporation gas' is controllable by the user according to the type
of mobile phase. For example, high boiling point eluents, such as water, require
higher evaporator gas flows than low boiling point solvents, such as
dichloromethane. This technology enables the PL-ELS 2100 Ice to evaporate water
at sub-ambient temperature (ca. 15°C).
Figure 2: General Schematic of an ELSD
The analyte particle exits the drift tube and enters an optical chamber where the
solute particles pass through a collimated light beam. The light source is typically a
halogen lamp or a monochromatic laser-emitting diode. The light scattered by the
particle is detected using a photomultiplier or photodiode, and the magnitude of the
scattered light is dependent on the size of the particle formed. The size of the
analyte particle is dependent on several factors, including sample concentration,
physical properties of the mobile phase, gas flow and sample volatility. Typical
detection limits lie in the range 1-50ng on-column, which can sometimes be 2-3
orders of magnitude lower than with UV detection.
ELSD response is non-linear because its signal is related to the absolute quantity of
the compound and independent of the analyte’s optical properties. Consequently, it
does not obey Beer’s Law. A simple logarithmic manipulation of the data will
produce a linear relationship. Some caution must, therefore, be exercised for
applications that demand a very low limit of detection or very high accuracy.
ELSD Theory
There are four main processes by which the path of electromagnetic radiation or light
can change direction when passing through a medium containing a suspended
particulate phase. These are shown in Figure 3.
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Figure 3: Scattering Mechanisms in an ELSD
The importance of each of these processes depends on the radius of the particle (r)
compared to the wavelength () of the incident light. Rayleigh scattering is
-2
predominant when r/ is < 5x10 . When particle dimensions are greater than /20,
they no longer behave as point sources, and Mie scattering becomes predominant.
Once particle size approaches the wavelength of incident light, then reflection and
refraction begin to prevail.
In order to decide which mechanism is predominantly responsible for the “scattering”
observed in the PL-ELS 2100/2100 Ice, an estimate of the size of the particles
involved compared to the wavelength of the incident light can be made:
D0 
585 
u 
where D0
na
  
 597

 
 1000Q 


 Qa 
=
=



u
=
=
=
=
Q
=
=
Qa
0.45
1.5
 na D 3 / na D 2
mean drop diameter
number of drops in the size
range with diameter D
liquid surface tension
liquid density
liquid viscosity
relative velocity between the gas
stream and the liquid stream
volumetric flow rate of liquid
volumetric flow rate of gas
The particulate size may be varied by altering the gas velocity, the eluent flow rate,
the temperature of the nebulizer and the initial solute concentration.
Changes in the solute concentration and variations in the atomizer gas pressure
influence the solute particle size. This relationship gives the instrument a maximum
sensitivity around r/ = 4. Detection declines rapidly when values for r/ are above 5
or below 2.5. When r/ < 2.5, the interference effects, typical of Mie scattering,
cause the deflected light to be low in intensity at the measuring angles. As the
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particles increase in size, reflection and refraction become dominant and sensitivity
increases. A further increase in the particle size causes the ratio of surface area to
volume to decrease, thus the sensitivity decreases.
The distribution tails as diameter increases, the largest particle in a distribution
generally reaching twice that of the mean. Consequently, although there is
undoubtedly some Mie and Rayleigh scattering, the observed phenomena are
predominantly due to reflection and refraction since the majority of the particles are
larger than the incident wavelength.
The relative importance of refraction and reflection can be understood by examining
the effects of the incident light on a single spherical particle whose equilateral axis
lies in the same plane as the photodetector and light source. With this configuration,
refraction is of greater significance than reflection. The majority of organic
compounds have refractive indices between 1.3 and 1.5. Changes in the refractive
index within this range will not greatly affect the quantity of light reaching the
detector. This accounts for similarities in the sensitivity of the instrument to various
compounds.
Therefore, the PL-ELS 2100/2100 Ice is useful as a universal detector, providing that
the material under investigation is non-volatile under the operating conditions of the
instrument.
Instrument Design
Nebulizer
The design and function of the nebulizer in the PL-ELS 2100/2100 Ice directly
influences all subsequent stages of operation, consequently the nebulizer design is
key to the performance of the PL-ELS 2100/2100 Ice.
Figure 4: PL-ELS 2100/2100 Ice Nebulizer Cross Section
The nebulization stream can be categorized into three distinct aerosols, namely
primary, secondary and tertiary. The primary aerosol describes the plume formed
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straight after the nebulizer tip, whereas the tertiary aerosol is the portion of
droplets/particles that reaches the detection cell (see Figure 4). Consequently, it is
the properties of the tertiary aerosol that are important for ELSD performance,
however, the properties of the tertiary aerosol are dependent on the characteristics of
the primary aerosol. The nebuliser chamber has a glass lining to prevent droplets
beading on the inner surface, which would otherwise cause excessive turbulence and
poor reproducibility.
Within the PL-ELS 2100/2100 Ice, the nebulizer gas flow is fixed at 0.4 SLM, as this
is the optimum gas flow for the operation for the nebulizer. Changing nebulizer gas to
reflect the mobile phase characteristics is not required with the PL-ELS 2100, as the
nebulizer is highly efficient. Low viscosity solvents are nebulized as effectively as
high viscosity solvents.
The PL-ELS 2100/2100 Ice nebulizer can be heated to 90°C, which can help in
reducing viscosity changes across a solvent gradient. Typically, the nebulizer
temperature is set to the same value as the evaporator temperature, although
nebulizer temperature has minimal effect on sensitivity.
Evaporator Design
PL-ELS 2100
The technique of ELSD relies on removing solvent by evaporation prior to detection.
For non-volatile compounds, this approach is straightforward, as the evaporator
temperature can be set at, or close to, the boiling point of the mobile phase. In order
to analyze semi-volatile species, the ELSD needs to operate at 30°C, which makes
the evaporation of solvents, such as water, problematic.
As Figure 4 shows, to dry a 10µm water droplet at 30°C takes 2.4 times longer than
at 50°C. Therefore, as you reduce the evaporation temperature, the evaporation
tube needs to be longer, in order to remove the solvent. If the droplet size is doubled
from 10µm to 20µm, the problem is exacerbated, because a 20µm droplet takes 4
times longer to evaporate than a 10µm droplet at 30°C.
The PL-ELS 2100 has the shortest evaporation tube on the market, therefore, to
remove solvents such as water at ambient temperature, dry nitrogen gas is added
during the evaporation step; this is referred to as “evaporation gas”. This evaporation
gas reduces the relative humidity (vapor loading) of the surrounding gas in the
evaporation tube, which in turn reduces the drying time. Figure 5 shows the effect
that evaporation gas has on the drying time of water droplets at 30°C. By reducing
the relative humidity from 70% to 50% for a 20µm droplet, the drying time is halved.
Therefore, the addition of gas during the evaporation step allows the evaporation
tube to be shorter, and facilitates low temperature operation at ambient temperature.
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Figure 4: Drying Time for Water Droplets at different Evaporator Temperatures
Figure 5: Drying Time for Water Droplets at 30°C under different Relative Humidities
The evaporation gas is a user defined parameter and adjusted according to the
mobile phase conditions.
PL-ELS 2100 Ice
The PL-ELS 2100 Ice operates in the same way as the PL-ELS 2100, the only
difference is the PL-ELS 2100 Ice can operate at sub-ambient temperatures.
.
The PL-ELS 2100 Ice comprises a peltier cooled/heated evaporation tube as shown
in figure 5. This allows the PL-ELS 2100/2100 Ice to operate at sub-ambient
temperatures, such that, the evaporation tube can be heated to 80° and cooled down
to 10°C, depending on the ambient temperature. The evaporator tube has a cool/heat
rate of ca. 4°C/min.
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Figure 7: PL-ELS 2100/2100 Ice peltier cooled/heated evaporator section
Optical Design
The optical bench of the PL-ELS 2100/2100 Ice has been specifically designed to
maximize sensitivity, while keeping the background noise to a minimum. The light
source is a short wavelength (480nm) Light Emitting Diode (LED), which provides
maximum sensitivity for small particles. The shorter the wavelength of light, the more
efficient the scattering process becomes because the smaller particles appear larger
to the incident radiation, hence refraction and reflection predominate.
The LED source is also a cool light source (unlike a halogen lamp), so internal
heating of the instrument, which would otherwise prevent low temperature operation
of the ELSD, does not occur. A feedback monitor maintains a constant output over
the lifetime of the ELSD.
The focal path length of the LED is such that the light beam covers the largest area
of the particle stream, consequently, the optics are ‘bent’ to accommodate this (see
Figure 8). To maximize detection of the scattered light, the photomultiplier (PMT)
detector module is mounted as close as possible to the scattering region. The PMT
is also arranged at the optimized angle of 120°C to the incoming radiation to ensure
maximum sensitivity. The PMT and LED on the PL-ELS 2100/2100 Ice have been
“matched” to the same wavelength (480m), because the PMT is at its optimum
sensitivity at this wavelength.
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Figure 8: Cross Section of Optical Bench in the PL-ELS 2100
To prevent condensation forming on the optical surfaces, the optical bench on the
PL-ELS 2100/2100 Ice is kept at a constant temperature of 50°C and is “swept” by
nitrogen gas at 0.4 SLM.
Digital Signal Processing/Filtering
The output of the PL-ELS 2100/2100 Ice is converted to a digital signal prior to the
analog output. Consequently, the PL-ELS 2100/2100 Ice offers two digital features as
outlined below.
Gain Function (PMT)
This parameter sets the factor by which the output signal is amplified by the detector.
The gain setting does not change the sensitivity of the detector. It merely amplifies
(or divides) the captured signal by the inputted factor (see figure 9). The gain can be
adjusted from 1 to 10, in increments of 0.1. When setting a PMT (or Gain) value,
both the signal and noise are simply amplified by the value set. The raw signal output
displayed on the parameter screen when the values are changed will reflect this
increase or decrease in signal amplification. However, the instrument output and the
output displayed on the main operating screen are automatically zeroed to 10mV
following a PMT change, and thus the recorded baseline position will remain
unchanged. The magnitude of the baseline noise will be the obvious indication of the
change in PMT.
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Figure 9: Example of Changing the PMT from 1 to 8
Smoothing Function (smth)
The smoothing function is defined as “the number of data points over which the data
is averaged”, and can be regarded as a digital time constant. For most applications,
the default value of 1 (0.1secs) is satisfactory, however for noisy or spiky data, then it
may be beneficial to average the raw data by increasing this smoothing parameter.
The maximum smoothing width that can be applied is 50 (5 secs). The value is
changed by increments of 1. Caution must be exercised when using this function, as
Figure 10 shows; the peak width can become wider if the smoothing function is set
too high.
Note: The data rate transmitted by the ELSD is a constant 10Hz, regardless of
the smoothing setting.
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ELS2100 DAC QC - SMOOTHING
300
Output (mV)
250
NO SMOOTHING
200
adca-00
5 SEC SMOOTHING
150
adca-00
100
50
0
0
20
40
60
80
100
Tim e (s)
Figure 10: Example of Smoothing Data
In addition to the digital features, the User is also permitted to change the power of
the LED source of the PL-ELS 2100/2100 Ice. The LED power enables the
brightness of the LED to be adjusted from 0% (off) to 100% (default). This feature is
extremely useful if the sensitivity of the detector needs to be reduced, bringing large
peaks back on scale (e.g. Preparative Chromatography).
Connections
The eluent from the chromatography system is connected to the front of the
instrument via the low dead volume Valco bulkhead connector provided (see Figure
11). The liquid inlet port is connected directly to the nebulizer by a short length
(190mm) of capillary tube, giving a delay volume from port to nebulizer tip of ~5µl.
The instrument should be supplied with clean, dry nitrogen gas at a head pressure of
60psi. A 4mm push-in connector is provided at the rear of the instrument for a
convenient connection to the gas source. To prevent unnecessary gas usage, an
automatic, but controlled, gas shut off valve is integrated into the gas inlet manifold.
This will only allow gas to pass into the instrument when the instrument is operating.
Should the instrument default to a standby mode, the gas valve will close after 15
minutes.
The PL-ELS 2100/2100 Ice is fitted with a standard RS232 (DTE-DCE) 3-wire serial
interface.
The serial RS232 connector provides a 24bit (10Hz) digital output for connection to a
chromatographic acquisition device. This facilitates data acquisition via a PC running
Galaxie™ Workstation
The PL-ELS 2100/2100 Ice can be controlled from a PC using the RS232 interface
and the PL-ELS 2100/2100 Ice graphical control software. If there are no available
serial ports on the PC, a serial to USB adapter can be used via the USB port on the
PC.
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For Internal Use Only
The I/O connector of the PL-ELS 2100/2100 Ice can be connected to auxiliary
equipment to pause or stop the operation of a pump or autosampler if the PL-ELS
2100/2100 Ice reports an error condition.
The PL-ELS 2100/2100 Ice is equipped with 2 contact closures, which are normally
open, and the available configurations are shown in Table 1.
Outputs
Inputs
I/O Description
User contact closure – normally
open
Pump stop contact closure –
normally open
TTL active low
TTL active high
Ground (to case)
Remote A/Z
Pin No.
4 & 12
3 & 10
2 & ground
9 & ground
1, 5, 6,11
7 & ground
[Firmware version 1.0.15. Units with serial
# 004-161 and before will also require a
wiring modification on the main PCB]
Table 1: Control I/O Connections
Detector Output
(1V)
Serial RS232
Connector –Digital
Output
Solvent Inlet
Firmware Flash Upgrade
Connector
I/O Connector
Gas Inlet
Liquid Waste Outlet
Figure 11: Front & Rear View of the PL-ELS 2100
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Detector Electronics
The PL-ELS 2100/2100 Ice is controlled using a single PCB with surface mounted
electronics. The main board controls all the functions of the detector. It contains
Flash RAM, which allows the firmware to be upgraded very quickly via the firmware
port on the rear of the detector. The main board also includes an internal vapor
sensor, a DAC and the PL-ELS 2100 Ice has a TEC control device.
The PL-ELS 2100/2100 Ice also include device identification, which allows multiple
ELSDs to be connected to one PC, e.g. Using Galaxie CDS. The Device ID is set at
1 in the factory, but is programmable in the field. Please refer to the service manual
for programming the Device ID.
Front Panel Control
The PL-ELS 2100/2100 Ice can be controlled through the graphical display on the
front of the instrument. Using this interface, you can select method, evaporator
temperature, nebulizer temperature, gas flow and digital features, using the
interactive menu bar at the bottom of the display. Table 2 briefly describes the
functions of each of the key commands.
Function

MODE
METHOD
EVAP
NEB
GAS
 or

Description
Options
Access to the parameter settings
menu
Modes of instrument operation
STANDBY
RUN
Displays and changes the instrument
DEFAULT
method
1-10 programmable methods*
Displays and changes the evaporator
OFF, 25-120°C (PL-ELS 2100)
temperature
OFF, 10-80°C (PL-ELS 2100 Ice)
Displays and changes the nebulizer
0-90°C (in 1°C increments)
temperature
Displays and changes the gas flow
0.90-3.25 SLM
Home (or lock) position displays actual Real time display can be
values and actions any changes made accessed by pressing the up and
in the menu options
down arrow keys when in this
The locked icon () will be displayed position.
with the instrument is controlled by the
PC based software
Table 2: PL-ELS 2100/2100 Ice Operating Functions
* The method editor software supplied with the instrument enables the methods to be
edited and stored on a PC and downloaded to the instrument.
The real-time output of the photomultiplier can also be displayed graphically via the
graphical interface. This provides the user with a means of monitoring detector output
without data acquisition. Access to this display is achieved by pressing the up/down
arrow keys while in home (lock) mode. Further  or  key presses changes the
display range.
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Modes of Operation
The PL-ELS 2100/2100 Ice can be operated in two modes, STANDBY and RUN,
both of which are described below.
STANDBY
The STANDBY mode is the “ground state” of the PL-ELS 2100/2100 Ice, which is
initiated automatically after power on. In this mode of operation, the heaters are off,
and the gas manifold valve is closed. The STANDBY mode gives the User a control
platform in which to setup the operational parameters (gas flow, nebulizer and
evaporator temperatures) before switching the unit into RUN mode. The instrument
will default to STANDBY mode should an error occur.
When the instrument is switched from RUN mode to STANDBY mode following a
command or error, then the gas management system is invoked and the gas set to
minimum flow of 1.2 SLM for 15 minutes before the gas manifold valve is closed.
This minimum “blanket” gas is enough to nebulizer and evacuate solvent should the
instrument default to STANDBY mode with solvent still flowing [firmware 1.1.8 and
greater].
RUN
The RUN mode is the normal operational mode. The instrument is now being
controlled at the set temperature and gas flow, and the system is fully operational.
During heating or cooling the instrument will display ‘NOT READY’ to show that the
system has not reached the set conditions. When the instrument has equilibrated,
‘READY’ will be displayed, and the instrument is ready for use.
Ten custom methods can be stored onboard for specific applications (see Table 3 for
the 10 default methods), but these cannot be edited via the keypad on the front of the
instrument. Custom methods can be created using the PL-ELS 2100/2100 Ice
method editor software, and subsequently downloaded onto the instrument.
Method
1
2
3
4
5
6
7
8
9
10
Evap (°C)
0
30
50
50
70
90
90
120
120
120
PL-ELS 2100
PL-ELS 2100 Ice
Neb (°C)
Gas (SLM)
Evap (°C)
Neb (°C)
Gas (SLM)
0
1.6
30
30
1.6
30
1.6
25
30
1.6
50
1.6
20
30
1.8
50
1.6
15
30
2.2
50
1.6
20
80
1.6
50
1.6
40
40
1.4
50
1.2
50
50
1.0
50
1.2
80
60
1.0
90
0.9
80
80
0.9
90
2.8
80
90
3.0
Table 3: PL-ELS 2100/2100 Ice Default Methods
Error Conditions
The PL-ELS 2100/2100 Ice is equipped with a number of sensors and error checking
facilities to ensure safe operation. If an error is detected, the instrument gives an
audible warning and a visible description of the error condition. In the event of an
error condition, the unit defaults into the STANDBY mode in which the heaters and
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gas are turned off. A complete list of instrument errors and remedial actions are
given in the troubleshooting section of this manual.
Once the source of the problem has been corrected, the error message will be
cleared automatically on the instrument display.
Computer Control
The PL-ELS 2100/2100 Ice can be controlled via a PC running the ELSD control
software (as supplied), or Galaxie workstation. The control software will automatically
adjust depending on the model of the ELSD connected to it.
Control Software
This Windows-based control software offers a second and complementary level to
total detector control. An intuitive single control panel provides control as well as a
comprehensive monitoring system.
Operational parameters can be easily
manipulated, saved or loaded by using the flexible Methods Editor, enabling rapid set
up and custom method archiving.
Control of the PL-ELS 2100/2100 Ice is through a simple control window. Within the
control window, the status of the detector is displayed, together with options to
control the various instrument parameters. The control window is effectively divided
into three main sections:
1. CONTROL
 View the current mode of operation
 Change the current mode of operation between Standby and Run
 Change the Current Method of operation between a stored custom method and
the default method ‘XXX’
 Change Parameters in the default method ‘XXX’
2. PARAMETER STATUS
Displays the actual values against the set parameters for:
 Evaporator Temperature
 Nebulizer Temperature
 Gas Flow
3. PHOTO DETECTOR
 Displays current detector output
 Displays current gain setting
 Remote Autozero
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Figure 12: PL-ELS 2100/2100 Ice Control Software
The PL-ELS 2100/2100 Ice control software offers all the features offered on the
instrument control panel, and thus the instrument settings, including gain adjust,
smoothing and LED intensity, can be adjusted via the Edit menu (EDIT / Instrument
settings menu option). Changes made to these parameters need to be downloaded
to the PL-ELS 2100/2100 Ice using the UPDATE button.
The PL-ELS 2100/2100 Ice control software allows the User to set up an automated
sequence of events during a 24-hour period. Using a time-triggered event approach
(based on the PC clock), the User can select a method of choice to be loaded and/or
the PL-ELS 2100/2100 Ice status changed.
System Test
The PL-ELS 2100/2100 Ice control software has an additional feature of System
Test, where the performance of the detector can be checked to ensure it is operating
to the required standard set at the factory. When the User selects this feature
(TOOLS / Run system test menu option), the software will run the detector through
a number of tests and analyse the information it gains, providing the User with an
analysis of the results.
Polymer Laboratories advises that this test is used occasionally to check the
performance of the instrument, or when a problem is encountered.
System Test
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The PL-ELS 2100/2100 Ice can store 10 customs methods, which can be selected
via the front of the instrument or via the control software. The Method Editor program
allows the User to create, edit and download these custom methods for method
development purposes or specific applications. The program can be launched from
the control software via this icon
Select method #
Edit the
control
parameters
Download to PL-ELS
2100
Add name
and
description
Figure 13: Method Editor window
Detector Performance
Detector Noise
Detector noise on the PL-ELS 2100/2100 Ice can originate from three sources:
electrical, gas and solvent. The electrical noise should not exceed 0.1mV, while the
gas noise should not exceed 0.2mV. If the solvent has been evaporated
successfully, the baseline noise with eluent flow should not exceed 0.3mV. However,
the presence of non-volatile additives or impurities in the solvent will dramatically
increase the baseline noise.
HPLC solvents should have a “residue after evaporation/ignition” value of
<1ppm.
The baseline noise can be smoothed using the smoothing function on the PL-ELS
2100/2100 Ice, as shown in Figure 14.
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Output (mV)
ELS2100 DAC QC - NOISE
41
40.5
40
39.5
39
38.5
38
37.5
37
36.5
36
400
Key:
PL-ELS 2100 smoothing setting:
10adca-0011
(1 sec)
30 (3 sec)
40adca-0012
(4 sec)
50adca-0013
(5 sec)
adca-0014
450
500
550
600
Tim e (s)
Figure 14: Effect of Smoothing Function on the Baseline Noise of the PL-ELS 2100
Sensitivity
The PL-ELS 2100/2100 Ice, as with other ELSDs, has a typical LOD range of 1-50ng
on-column.
For the PL-ELS 2100, the LOD of caffeine is 19ng or for glucose is 50ng oncolumn.
However, caution must be exercised when comparing limits of detection between
ELSDs. The sensitivity of any ELSD is compound specific depending on the type of
mobile phase, flow rate and design of the ELSD. For non-volatile compounds,
optimum sensitivity is achieved at high temperature, while for semi-volatile
compounds, low temperature operation is required to minimize loss through
volatilization.
In addition, direct injections can be performed on ELSDs and sometimes an LOD
may be quoted which refers to the direct injection measurement.
The only true way of comparing ELSDs is by a head-to-head comparison, and even
then, the operating temperatures of the ELSD must be correctly optimized to achieve
the highest sensitivity. It is for this reason that most ELSDs are sold by
demonstration.
Linearity
ELSD is a non-linear technique, due to the four main scattering processes:
1.
2.
3.
4.
Rayleigh Scattering
Mie Scattering
Reflection
Refraction
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The dominance of each of these processes depends on the radius of the analyte
particle (r) compared to the wavelength () of the incident light. However, the
intensity of these four scattering mechanisms is not equal. Therefore, at high
concentrations, particle size is large and refraction and reflection dominate, hence
the response is linear in fashion. However, when the concentration of the analyte is
lowered, the size of the analyte particle gets smaller, and weaker scattering
mechanisms of Mie and Rayleigh dominate. The consequence of this disparity in
scattering strength gives the evaporative light scattering detector a non-linear
response, especially at low concentrations (see Figure 15).
Figure 15: Scattering Mechanisms in ELSD and their Effect on Response
The analyte concentration and peak area response for an ELSD are related by the
following expression:
A = amb
Where, A is the measured peak area, m is the sample mass and a and b are
coefficients depending on droplet size, concentration, the nature of the solute, gas
and liquid flow, as well as vaporization temperature. By plotting concentration vs
peak area for this relationship, a curved line is obtained. The chart in Figure 16
shows data plotted on a standard axis. A linear trend line does not fit the data well
(R2=0.9902), but a quadratic expression gives R2 as 0.995.
Page 18 of 24
For Internal Use Only
Comparison of Trendlines fits for Customer data
600000
Linear (Series1)
Power (Series1)
500000
y = 1785.5x1.4702
R2 = 0.9948
y = 12268x - 83991
R2 = 0.9902
peak area
400000
300000
200000
100000
0
0
10
20
30
40
50
60
Concentration
Figure 16: Concentration vs Peak Area for the PL-ELS 2100/2100 Ice response
For ease of manipulation, all ELSD users employ the logarithm of equation 1 to give
the following expression:
log
A = b log m + log a
Log-Log Plot of Customer data
6
5.8
y = 1.4702x + 3.2518
R2 = 0.9948
5.6
log peak area
5.4
5.2
5
4.8
4.6
4.4
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
log Concentration
Figure 17: ELSD data from figure 15 expressed with Log Axes
Therefore, by plotting the log concentration vs the log peak area, the more familiar
linear fit can be applied to the data (see Figure 17).
The value of b has been shown to be highly dependent on the mobile phase eluents
and temperature, with typical values ranging from 0.8-1.2. Calibration data is not
forced through the origin, as the detector will give a small response at zero
concentration due to fluctuations in electrical and gas noise.
Page 19 of 24
For Internal Use Only
Uniformity of Response
The majority of pharmaceutical compounds possess a UV chromophore, hence UV
detection is the first choice detection method. However, the use of a UV detector can
limit the sensitivity and efficiency of a separation as the optimum detection
wavelength may not be compatible with the mobile phase eluent. In addition,
different responses are obtained for different species at the same concentration due
to differences in their extinction coefficient.
By contrast, since all analyte particles scatter light, the ELSD can detect all samples
with high sensitivity and accuracy, irrespective of their chemistry or optical properties.
Consequently, when developing chromatographic methods for active ingredients
using ELSD, the analyst can select the appropriate solvent system to give the
optimum separation without compromising the sensitivity or resolution. ELSD also
provides a uniform response to all components, as highlighted in figure 18 for aspirin
and phenacetin. A solution containing equal quantities of aspirin and phenacetin
produces an equal response for both compounds using the PL-ELS 2100, whereas
UV detection exhibits a lower response for aspirin. This is due to a difference in
extinction coefficient between the two compounds. This example highlights the
advantage of using ELSD when analyzing mixtures of pharmaceutical compounds.
Figure 18: Comparison of the Uniformity of Response between ELSD and UV/Vis
for Analysis of Aspirin and Phenacetin at Equal Concentrations
Dispersion
Evaluation of peak dispersion of the PL-ELS 2100/2100 Ice for caffeine was
undertaken at 3 eluent flow rates. A PL-ELS 2100/2100 Ice and Sedex 75 ELSD
were evaluated at flow rates of 0.5ml/min, 0.25ml/min & 0.1ml/min. Figure 19 shows
the plate counts measured at each flow rate for the two detectors. The PL-ELS
2100/2100 Ice was evaluated with both a 0.007” and 0.010” ID nebulizer needle. The
PL-ELS 2100/2100 Ice uses a 0.010” ID needle for nebulization as the 0.007” ID
needle is prone to blocking.
Page 20 of 24
For Internal Use Only
Plate counts vs Flow rate
35000.00
30000.00
Unit #049 (10 thou)
Unit #058 (7 thou)
SEDEX 75 (HPLC)
Plate counts N (m)
25000.00
20000.00
15000.00
10000.00
5000.00
0.00
0
0.1
0.2
0.3
0.4
0.5
0.6
Flow rate (ml/min)
Figure 19: Dispersion of PL-ELS 2100/2100 Ice for different Nebulizer Needles
PL-ELS 2100 Ice Heating/Cooling performance
The PL-ELS 2100 ICE can be heated or cooled very rapidly in order to switch
between ELSD methods. Figure 20 shows the cooling rate of the PL-ELS 2100 ICE
PL-ELS 2100 Cooling curve
from 50-30°C.
50-30°C
50
Temp (°C)
45
40
35
30
0.0
1.0
2.0
3.0
4.0
Time (min)
Figure 20: PL-ELS 2100 Ice evaporator cooling curve
The PL-ELS 2100 Ice has a heating/cooling rate of ca. 4°C/min, however the
performance of the peltier unit is dependent on ambient temperature. If the ambient
temperature is >25°C, then the PL-ELS 2100 Ice can only achieve a minimum
temperature of 15°C.Whereas at an ambient temperature of <25°C, a minimum
evaporator temperature of 10°C can be achieved.
Page 21 of 24
5.0
For Internal Use Only
Best Practice for the PL-ELS 2100/2100 Ice ELSD
Optimization
To achieve optimal performance from the instrument, certain operational conditions
should be met. An understanding of the nebulization/evaporation process will aid in
selecting the best operational parameters for that application. In theory, optimal
performance is achieved by producing a specific solute droplet size with a narrow
size distribution. The nebulized droplet size is affected by adjusting the eluent flow
rate, evaporation temperature and gas flow rate.
The effect of eluent flow rate on the instrument operation is dependent on the volume
of eluent that must be volatilized and on the droplet particle size. A higher eluent
volume may require a higher evaporation temperature, a higher gas flow rate, or
both, to achieve optimal conditions. The PL-ELS 2100/2100 Ice is designed to
receive eluent flow rates up to 5ml/min at ambient temperatures, even for 100%
water. However, at sub-ambient temperatures, eluent flow rates should be limited to
1ml/min for optimum performance
It is recommended that the instrument be operated at the lowest evaporator
temperature required for complete evaporation of the eluent. Optimal temperature
settings will be dependent on the volatility of the analyte and not the just the eluent.
Only volatile eluents should be used. Non-volatile eluent components (such as nonvolatile salts) will not evaporate and may collect inside the instrument, requiring
removal and cleaning of the evaporator tube.
The gas flow rate is the most varied parameter when optimizing performance. The
detector operates using clean, dry nitrogen at flow rates up to 3.25 SLM.
Adjustments to the gas flow rate can have a marked effect on detector response.
The maximum response is achieved when reflection and refraction increase as the
particle size approaches the optimum diameter.
For method development purposes, the nebulizer and evaporator temperatures of the
PL-ELS 2100/2100 Ice should initially be set at 30°C/15°C, with a gas flow of
1.6SLM/2.4 SLM. An injection of the analyte should be made and the response
recorded. A second injection should be made at slightly higher temperature (eg
40°C/20°C, gas 1.4/1.8 SLM), and a third injection at 80/50°C (gas 1.2 SLM). In
doing this, the optimum conditions of the PL-ELS 2100/2100 Ice will be obtained
when the highest response of the analyte is observed. The gas flow setting changes
the amount of gas passing up the evaporation tube and is adjusted according to the
volatility of the mobile phase. As the temperature is increased, the gas flow value
can be lowered. Excess evaporation gas will reduce the sensitivity of the detector,
therefore, the gas parameter should be operated as low as possible on the PL-ELS
2100/2100 Ice.
It is recommended to run the PL-ELS 2100 Ice at low flow rates (eg <1ml/min) if
possible, because sub-ambient evaporation is more efficient at these lower flow rates
and sensitivity is improved.
Chromatography Requirements
The PL-ELS 2100/2100 Ice requires the use of volatile mobile phases and additives
in order to function correctly. The PL-ELS 2100/2100 Ice is compatible with most
Page 22 of 24
For Internal Use Only
HPLC mobile phases, with the exception of DMF & DMSO. Sodium or potassium
salts and mineral acids are not compatible with the ELSD. Those additives that are
compatible with ELSD are shown in Table 4.
pKa
pKb
Acids
Trifluoroacetic acid
Formic acid
Acetic acid
Carbonic acid
0.3
3.75
4.75
6.37
13.70
10.25
9.25
7.63
72.4°C
100.7°C
116.0°C
-
Bases
Ammonia
Methylamine
Ethylamine
Triethylamine
9.25
10.81
10.66
11.01
4.75
3.19
3.34
2.99
-33.35°C
16.6°C
-6.3°C
89.3°C
Buffers
Ammonium formate
Pyridinium formate
Ammonium acetate
Ammonium carbonate
pH range
BP
3.0-5.0
3.0-5.0
3.8-5.8
5.5-7.5 and 9.3-11.3
Ion Pair Reagents
Pentafluoropropionic acid
~0.6
Heptafluorobutyric Acid
~0.6
Nonafluoropropionic acid
~0.6
Pentadecafluorooctanoic acid
~0.6
Tridecafluoroheptanoic acid
~0.6
Table 4: ELSD Compatible Mobile Phase Additives
MP
120°C
111°C
97°C
120°C
140°C
189°C
175°C
Maintenance
There is a potential for impurities to accumulate in the evaporation tube and nebulizer
due to the nature of the instrument. Consequently, it is highly recommended that the
detector is cleaned on a regular basis to prevent this build up of contamination. The
simplest way to prevent fouling of the instrument is to include a wash cycle in a
sequence of injections, or to ensure that clean mobile phase is flushed through the
system prior to switching the unit off.
If the instrument does become contaminated, a “steam clean” can be performed
whereby the instrument is heated to maximum temperature and the gas flow is set to
3.0 SLM. Water is then passed through the instrument for 16 hours (or overnight) to
remove the contamination. Alternatively, the instrument can be flushed with a
suitable solvent, if water isn’t applicable, for the same period of time.
Page 23 of 24
For Internal Use Only
Specifications
Light Source
Detector
Temperature
Range
Gas requirements
Blue LED 480nm
Photomultiplier tube with additional digital
signal processing
Evaporator
PL-ELS 2100
PL-ELS 2100 Ice
Nebulizer
Flow rate
Pressure operating
range
Maximum Pressure
Eluent Flow rate
Analogue Output
Digital Output
Communication
Outputs
Input
Instrument
Operation
Power
Requirements
Detector Status
Size
Weight
Unpackaged
Packaged
Unpackaged
Packaged
ambient-120°C (1°C increments)
10-80°C (1°C increments)
Ambient-90°C (1°C increments)
Up to 3.25 SLM @ 60 psi @25°C
With integrated automatic, controlled gas
shut-off valve
60 – 100 psi (4-6.7 bar)
100 psi (6.7 bar)
0-5 ml/min
0-1V FSD
24bit digital data, 10Hz via serial port
Serial I/O (RS232)
1 User Contact closure
Pump stop: 1 contact closure
1 TTL +ve
1 TTL –ve
Auto zero
Graphical Vacuum Fluorescent display
5 button keypad
10 predefined methods
PC based method utility program
90/120V AC or 220/250V AC 50/60 Hz 2A
max
Standby, Run
200x450x415 mm (wxdxh)
360x700x600mm (wxdxh)
11kg
16 kg
Page 24 of 24