Evaporative Light Scattering Detectors

from Polymer Laboratories

 Introduction

ƒ Polymer Laboratories has over 15 years of experience in ELSD

ƒ ELSD can outperform traditional detectors when analysing non-chromophoric
samples by HPLC
ƒ Traditional HPLC detectors such as UV and RI have limited capabilities:
ƒ UV and RI are not compatible with a wide range of solvents
ƒ RI detection is not gradient compatible
ƒ Different analytes produce different UV responses depending on their
extinction co-efficient
ƒ ELSDs can detect anything that is less volatile than the mobile phase
ƒ ELSD is universal and compatible with a wide range of solvents
 Introduction

Polymer Laboratories presents 3 models of ELSD, each offering high sensitivity for
a wide range of applications:

PL-ELS 2100
ƒ Regular HPLC with low temperature operation for volatile compounds

PL-ELS 1000
ƒ Routine HPLC and GPC using high boiling point solvents

PL-ELS 1000µ
ƒ For microbore and capillary LC
New PL-ELS 2100 Detector
 Principles of Operation

The ELSD principle of operation employs three distinct stages:

ƒ Nebulisation
ƒ Evaporation
ƒ Detection
Gas

Eluent inlet

Light Source
Liquid waste
 Nebulisation

ƒ Eluent flow mixed with N2 or Air

ƒ Concentric nebuliser
ƒ Efficient nebulisation: stable droplet plume,
uniform droplet size
ƒ Temperature independently controlled
Narrower cone of the plume
(minimise band broadening)
ƒ Small nebuliser chamber
(reduces band broadening)
 Evaporation

ƒ Droplets pass through heated drift tube

ƒ 30cm straight tube
ƒ Removes mobile phase to leave
particulate form of analyte
ƒ Temperature set according to analyte
volatility
ƒ Temperature controlled by user
ƒ Important to have laminar flow
(reduces band broadening)
 Detection

ƒ Particles irradiated with light source

ƒLED (ca 400-500 nm)
ƒ Particles scatter light according to their size
(mass sensitive)
ƒ Scattered light is detected by photomultiplier
at fixed angle from incident light
ƒ Detection independent of optical properties
of analyte
 Advantages of
Evaporative Light Scattering Detection
ƒ Universal - responds to all compounds in the mobile phase

ƒ Not dependent on spectroscopic properties of analyte

ƒ Produces more uniform detection sensitivity for analytes

ƒ Not susceptible to baseline drift during gradient elution, temperature or solvent

pump fluctuations

ƒ ELSD compatible with a much wider range of solvents compared to Refractive

Index
 Advantages of
Evaporative Light Scattering Detection

ƒ Removes the need for derivatisation steps (eg amino acids, toxins)
ƒ Fast setup and equilibration
ƒ Sensitivity in the order of 1-50ng (on column)
(depends on eluent flow rate)
ƒ No interference from solvent front peaks
(enables fast analysis)
ƒ Removal of mobile phase eluent allows rapid HPLC gradients
ƒ Flow rates up to up to 5ml/min can be achieved with no affect on baseline
stability
ƒ Ideal for High Throughput Screening
Fast Gradient, Fast Flow Rate Capability

Column: PLRP-S 100Å 5µm, 50x4.6mm

Eluent A: Water + 0.05% TFA
Eluent B: ACN + 0.05% TFA
Gradient: 5-95% B in 1 min
Detector: PL-ELS 2100
(neb=30°C, evap=30°C, gas=1.6 SLM)

Sample: Indapamide(IND), Dibutyl phthalate (DBP)

Flow rate increased from 2ml/min up to 5ml/min

Note: IND is non-volatile, DBP is relatively volatile

 Fast Gradient, Fast Flow Rate Capability
0 DBP
IND
4
2ml/min

8
3ml/min

2
4ml/min

5ml/min
0
0 1 2 3
Retention time (min)
 Fast Gradient, Fast Flow Rate Capability
Stable baseline through the gradient

2ml/min

3ml/min

4ml/min

5ml/min

0.0 0.6 1.2 1.8 2.4 3.0

 High Throughput Screening
PL-ELS 2100 is transparent to DMSO at ambient temperature !!

Eluent A: 0.1% formic acid in water/ACN 95:5 v/v

Eluent B: 0.1% formic cid in water/ACN 5:95 v/v
Gradient: linear 0 - 100% B in 5 min
Flow Rate: 0.9 mL/min
Samples: 1 ng on column
Detector: ELS 1000

Nebuliser 30°C Evaporator30°C

(peak 2 DMSO )
 Evaporative Light Scattering Detection
Ideal Complement to LC-MS

ƒ Similar operating principles to LC-MS

ƒ Volatile buffers
ƒ Favours lower flow rates (ie 0.2-0.5ml/min)

ƒ Can develop LC methods on ELSD then transfer to LC-MS

ƒ ELSD can provide supporting information when used in tandem with LC-MS
 Ideal Complement to LC-MS
Sample Mixture of known 1:1 ratio

LC-MS
results show ratio to be
3:1

UV-Vis
results show ratio to be
10:1

PL-ELS 2100
results show ratio to be
1:1
(Response independent
of optical properties)
 Operation of the PL-ELS 2100
For Volatile & Semi-Volatile Solutes

ƒ A new approach to ELSD applications

ƒ Nebuliser and evaporator temperatures
determined by the volatility of the ANALYTE
ƒ Gas flow used to aid evaporation of the mobile
phase
ƒ 100% water @30°C requires high gas flow
ƒ 100% Hexane @30°C requires no gas flow
ƒ PL-ELS 2100 can operate in 100% water
@ 25°C
 Non-Volatile Application: Sugars
Column: PL Hi-Plex Ca 9µm, 300x7.7mm
Eluent: Water
Flow Rate: 0.6ml/min
Temp: 85°C
Inj Vol: 10µl
Detector: PL-ELS 2100 (neb=30°C, evap=90°C, gas=1.6 SLM)
Samples: 1. Fructose, 2. Glucose, 3. Sucrose I, 4. Lactose, 5. Stachyose
3

1 5
2

0 min 30
Non-Volatile Application: Phospholipid Separation
Column: Lichrospher DIOL 5µm,150x2.1mm
Eluent A: IPA/Hexane/Water/Ammonia Hydroxide
57.8/40/2/0.2
Eluent B: IPA/Hexane/Water/Ammonia Hydroxide
51.8/40/8/0.2
Gradient: 0-100% B in 7 mins (hold 8 mins)
100-0% B in 5 mins (hold 10mins)
Flow Rate: 0.3ml/min
Detector: PL-ELS 2100
(neb=30°C, evap=80°C, gas=1.0 SLM)

Samples: 1. Cholesterol
2. Phosphatidylethanolamine
3. Phosphatidylcholine
4. Sphingomyelin
5. Lysophosphatidylcholine
 Semi-Volatile Application:
Underivatised Amino Acids

Column: Thermo-Hypersil ODS 5µm, 250x4.6mm

Eluent A: Water
Eluent B: Acetonitrile
Gradient: 100% A in 5 mins hold, 0-40% B in 20 mins
Flow Rate: 0.6ml/min
Inj Vol: 10µl
Detector: PL-ELS 2100
(neb=50°C, evap=50°C, gas=1.6 SLM)
 Semi-Volatile Application:
Underivatised Amino Acids
ELSD removes the need for derivatisation for applications such as amino acids

1. Serine
2. Glutamic Acid
3. Arginine
4. Proline
5. Valine
6. Methionine
7. Isoleucine
8. Leucine
9. Phenylanine
10. Tryptophan

0 min 30
 Volatile Application:
Effect of Changing Temperature
Column: Adsorbosil C18 5µm, 150x4.6mm
Eluent A: Water + 0.1% TFA
Eluent B: ACN + 0.1% TFA
Gradient: 60-90% B in 5 mins
Flow Rate: 1.0ml/min
Detector: PL-ELS 2100
(neb/evap=same temperature, gas=1.8 SLM)
Samples: 1. Acetanilide
2. Indapamide
3. Ibuprofen
4. Dibutylphthalate

Note: Peak 2 is non-volatile

Peaks 1, 3 & 4 are relatively volatile
 Volatile Application:
Effect of Changing Temperature
• At high temperature, peak 2 has better S/N but peaks 1,3 & 4 are not detected
• Running cold, all four peaks are detected

50°C

35°C

2 3 4

1
Ambient

0 9
Retention Time (min)
Volatile Application: Phthalate Separation
Column: X-Terra C18 3.5µm, 30x2.1mm Samples
Eluent A: Water 1. Diethylphthalate
Eluent B: ACN 2. Dipropylphthalate
Gradient: 0-100% B in 3 mins (hold 0.7 min) 3. Dibutylphthalate
Flow Rate: 0.5ml/min 4. Dipentylphthalate
Detector: PL-ELS 2100 5. Dioctylphthalate
(neb=25°C, evap=25°C, gas=1.6 SLM)
Volatile Application: Parabens Separation
Column: Adsorbosil C18 5µm, 150x4.6mm Samples
Eluent A: Water 1. Methylparaben
Eluent B: ACN 2. Ethylparaben
Gradient: 50-75% B in 7 mins 3. Propylparaben
Flow Rate: 1.0ml/min
Detector: PL-ELS 2100
(Evap=30°C, Gas=1.0 SLM)

Nebuliser 90°C

Nebuliser 30°C
 PL-ELS 2100
Detection of Low Molecular Weight Compounds
Column: Adsorbosil C18 5µm, 150x4.6mm
Eluent: 60/40 Water/Acetonitrile
Flow Rate: 1.0ml/min
Inj Vol: 10µl
Detector: PL-ELS 2100
(neb=30°C, evap=30°C, gas=1.6 SLM)
Sample: Urea 1mg/ml

PL-ELS 2100 can detect Urea,

which has a MW 60

0 min 4
 PL-ELS 2100
Sensitivity: Limits of Detection
Column: Spherisorb C18 5µm,150x4.6mm
Eluent A: 99% Water + 0.1% TFA
Eluent B: 1% ACN + 0.1% TFA
Flow Rate: 0.5ml/min
Detector: PL-ELS 2100
(neb=50°C, evap=70°C, gas=1.2 SLM)
Sample: DL-Glutamic Acid
 PL-ELS 2100
Sensitivity: Limits of Detection

Column: ACE C8 3µm, 50x4.6mm

Eluent A: 100% Water
Eluent B: 100% ACN
Isocratic: 50/50 A/B
Flow Rate: 1.0ml/min
Detector: PL-ELS 2100
(neb=50°C, evap=50°C, gas=1.4 SLM)
Sample: Caffeine
 Additional Benefits
of the PL-ELS 2100

ƒ Low temperature operation even for 100% water

ƒ Extremely low dispersion for high resolution separations
ƒ High eluent flow rates, up to 5ml/min
ƒ Improved uniformity of response across a solvent gradient
ƒ Rapid equilibration
ƒ Extremely small footprint, stackable
ƒ Ergonomic design
ƒ Easy to use
Les produits de nos partenaires
Système LC ECD ALexys
 Système LC ECD ALexys

Système intégré dédié à la détection électrochimique:

ƒFlexibilté : existe en version LC ou µLC
ƒSensibilité:
ƒEnsemble du système optimisé pour la détection électrochimique
ƒCompatibilité CFR 21 Par 11
Composants du système :
•Pompe, amortisseur de pulsations
•Dégazeur
•Injecteur automatique analytique ou micro
•DECADE II
•Système de traitement de données
 Decade II
Véritable plate forme de travail
• Un four, Cage de faraday, abritant cellule
de détection et colonne
• Pouvant contrôler une ou deux cellules de
détection ( mode série ou parallèle)
• Concept de cellule original ( vaste choix
d’électrodes de travail et de référence)

• Filtrage efficace du bruit de fond (ADF)

 Cellule ampérométrique VT-03

• Cellule de type Wall Jet

• Stabilisation rapide,
• Faible volume(0,3µL à 10nL)
• Sensibilité

A: couche mince, B wall jet, C Poreuse

ADF avdanced digital filtration
Filtre conçu spécialement pour la détection
électrochimique
NA - A - DHBA - D - 5HTRY - 5HT
0.06 10Hz
0.005Hz
0.05

0.04

0.03
I (nA)

0.02

0.01

-0.01
0 2 4 6 8 10 12 14 16 18 20
t (min)

Les basses fréquences ( pics) passent

Les fréquences élevées (bruit) sont bloquées
 Applications types

• Analyse de traces nécessitant: séléctivité et

sensibilité

• Applications en Micro dialyse ( Off line ou On

line)
 Micro dialysate (frontal cortex + nucleus accumbens) settings

ƒALEXYS system
ƒflow 1 ml/min; injection 25 ul (ul pick-up mode)
ƒcolumn 10 x 0.46 cm 3 um ODS-2; spiked with 10 fmol DA + 5HT/25ul (= 4 x 10-
10 M)

ƒADF 0.01 Hz up to 10 min, thereafter 0.002 Hz