Evaporative Light Scattering Detector (ELSD)

Size: px

Start display at page:

Download "Evaporative Light Scattering Detector (ELSD)"

Evan Stephens
5 years ago
Views:

1 BÜCHI Labortechnik AG Evaporative Light Scattering Detector (ELSD) Martin Verstegen ELSD 3300 page 1

2 Overview of Common HPLC Detectors 1. UV/Vis 2. RI 3. Fluorescence 4. Mass Spectrometry 5. ELSD page 2

3 UV/Vis Detectors Advantages Easy to use Reliable Relatively inexpensive Solvent gradient compatible Nondestructive to sample Relatively sensitive Specific Limitations Poor response if compound doesn t have a good chromophore group Solvents limited by UV cutoff especially at low UV page 3

4 RI Detection Advantages Universal nature of the detector response Good linear dynamic range - ~4 orders of magnitude Easy to operate Limitations Not particularly low limit of detection Very sensitive to temperature and pressure fluctuations Slow to equilibrate Get both positive and negative peaks Cannot be used with solvent gradients Better to premix solvents instead of letting a pump do the mixing page 4

5 Fluorescence Detection Advantages Very sensitive Very selective Generally insensitive to flow and temperature changes Limitations Limited linearity Complicated to use must have firm grasp on both chemical and instrument variables Some chemicals like oxygen may quench fluorescence must degas well Not many compounds naturally fluoresce Derivatization complicates method page 5

6 Mass Spectrometry Advantages Very sensitive Very selective Provides structural information Limitations Purchase price is very high Complicated operation Frequent maintenance page 6

7 Evaporative Light Scattering Detection (ELSD) History Detection principles Limitations Advantages Benefits for HPLC analysis page 7

8 Evaporative Light Scattering Detector History First commercialized in the early 1980s Most early work centered on Polymers By the early 1990s, used for a diverse range of applications Now, ELSDs are used by virtually every industry page 8

9 Unique Method of Detection Three Steps Nebulization Mobile Phase Evaporation Detection page 9

10 How Does ELS Detection Work? Step 1: Nebulization Column effluent mixes with gas flow to form a dispersion of droplets Step 2: Mobile Phase Evaporation Mobile phase evaporates from around sample particle leaving dried particles in solvent vapor Step 3: Detection Sample particles pass through laser light in a flow cell. The particles scatter light that is collected by a photodiode ELSD detects any compound less volatile than mobile phase page 10

11 How Does ELS Detection Work? Benefits of ELS Detection In Flash Chromatography Allows purification of nonchromophoric molecules Allows you to see impurities not seen by UV Gives a more accurate indication of relative quantities than UV Give you the freedom to choose UV absorbing solvents ELSD detects any compound less volatile than mobile phase page 11

12 From Nebulization to Detection See how the complete process works in the new 3300 ELSD page 12

13 ELSD Advantages Near Universal Detect any compound less volatile than the mobile phase Sensitive Low nanogram detection limits Easy to Use Uncomplicated operation Gradient Compatible Maintain stable baselines during steep gradients page 13

14 ELSD Limitations 1. Only volatile mobile phases can be used 2. The sample must be less volatile than the mobile phase 3. S-shaped response curve ELSD is a destructive technique, like MS last detector in line or use a splitter page 14

ELSD s Ability to Evaporate Mobile Phase at Near Ambient Temperatures Allows Detection of Semi-volatiles 10268 1.

15 ELSD s Ability to Evaporate Mobile Phase at Near Ambient Temperatures Allows Detection of Semi-volatiles Glycerol Column: Alltech Prevail Carbohydrate ES, 5µm, 250 x 4.6mm Mobile Phase: Acetonitrile: 0.04% Ammonium Hydroxide (80:20) Flowrate: 1.0mL/min page 15

16 Volatile Mobile Phase Modifiers Acids pka pkb ph Range BP MP Trifluoroacetic Acid C Formic Acid C Acetic Acid C Carbonic Acid Bases Ammonia C Methylamine C Ethylamine C Triethylamine C Buffers Ammonium Formate Pyridinium Formate Ammonium Acetate C Pyridinium Acetate Ammonium Carbonate (for reverse phase) Ammonium Carbonate 8.0 (adjusted) and C page 16

17 Advantages of ELSD Compared to Other Detectors UV/Vis RI Fluorescence MS page 17

18 Benefits of ELSD vs. UV/VIS Benefits of ELSD Compared to UV/VIS Detector UV Detector Limitations: Only detects compounds that absorb UV or visible radiation Detection based on extinction coefficient ELSD Benefits: Detects any compound less volatile than the mobile phase Near equivalent response Baseline drift with gradients at low wavelength Solvent front interference at low wavelength Little or no baseline drift during gradients No response from solvents Solvent limited by UV cut-off Compatible with all volatile mobile phases page 18

19 ELSD is More Sensitive and Stable than Low-wavelength UV Steroid Conjugates Estrone 3-Sulphate, 100ng 2. β-estradiol 17-(β-D-Glucuronide), 100ng 3. 5β-pregnane-3α, 20α-diol Glucuronide, 220ng Column: 2.1mm Mobile Phase: Alltech Alltima C18, 5µm, 150 x (Part No ) A: 0.1% TFA in Water B: 0.1% TFA in Methanol Gradient: Time: %B: Flow Rate: 0.2mL/min page 19

ELSD Shows What May Be Missing from Your UV Chromatogram Lotrimin AF Topical Solution 10446 10447 1. PEG 2.

20 ELSD Shows What May Be Missing from Your UV Chromatogram Lotrimin AF Topical Solution PEG 2. Clotrimazole Column: Alltech Alltima C18, 5µm, 150 x 4.6mm (Part No ) Mobile Phase: A: Water B: Methanol Gradient: Time: %B: Flow Rate: 1.0mL/min Column Temp.: 40 C page 20

21 ELSD Obtains a More Accurate Representation of Sample Mass than UV 1:1 Mixture Ephedrine 2. Caffeine Column: Alltech Alltima HP EPS C18, 5µm, 150 x 4.6mm (Part No ) Mobile Phase: 1% Acetic Acid:Methanol:Acetonitrile (70:20:10) Flow Rate: 1.0mL/min page 21

22 Benefits of ELSD vs. RI RI Detector Limitations: Baseline instability due to temperature changes Negative and positive peaks in one run ELSD Benefits: Not affected by ambient temperature changes Always a positive response Gradient elution not possible Compatible with gradients Solvent front interference No response from solvents Poor sensitivity Nanogram-level sensitivity page 22

23 The ELSD Improves Baseline Stability and Detection Sensitivity Compared to RI Carbohydrates Fructose 2. Glucose 3. Sucrose RI ELSD Column: Alltech Prevail Carbohydrate ES, 5µm, 53 x 7mm (Part No ) Mobile Phase: Acetonitrile:Water (75:25) Flowrate: 2.0mL/min Column Temp: 30 C page 23

24 Fluorescence Detector Limitations Fluorescence Limitations: Usually requires pre or postcolumn derivatization ELSD Benefits: No derivatization necessary page 24

25 ELSD Eliminates Derivatization of Amino Acids Underivatized Amino Acids Gly 2. Ser/Asn 3. Asp 4. Gln 5. Ala/Thr 6. Glu 7. Cys/Lys 8. His 9. Pro 10. Arg 11. Val 12. Met 13. Tyr 14. Ile 15. Leu 16. Phe 17. Trp Column: Alltech Prevail C18, 5µm, 250 x 4.6mm (Part No ) Mobile Phase: A: 5mM Heptafluorobutyric Acid, ph1.0 w/0.7% TFA B: Acetonitrile Gradient: Time: %B: Flow Rate: 1.0mL/min page 25

26 Although the Mass Spectrometer is a Universal Detector, ELSDs Offer Many Benefits Over MS 1. Lower investment and operating costs 2. Less complicated operation 3. Less maintenance 4. More accurate quantitative results Since chromatographic requirements for ELSD and MS are similar, methods developed for ELSD are usually transferable to MS without modification. Develop MS methods offline and save the MS for real sample analysis. page 26

27 Use ELSD in Parallel with UV/Vis and MS for Maximum Structural and Concentration Information 1. Aspartame 2. Hydrocortisone 3. Reserpine 4. Terfenadine Chromatograms courtesy of Agilent Technologies Column: ZORBAX StableBond C18, 3.5µm, 50 x 4.6mm Mobile Phase: A: 0.025% Formic Acid in Water B: 0.025% Formic Acid in Acetonitrile Gradient: Time: %B: Flow Rate: 1.0mL/min Column Temp: 30 C page 27

28 The ELSD Does Not Respond to Changes in Mobile Phase Composition, Allowing Rapid Separations Using Extreme Gradients Ephedrine 2. Acetylsalicylic acid 3. Diphenhydramine 4. Trimipramine Column: Alltech Alltima HP C18, 3µm, 33 x 7.0mm (Part No ) Mobile Phase: A: 0.1% TFA in Water B: 0.1% TFA in Acetonitrile Gradient: Time: 0 3 %B: Flow Rate: 2.5mL/min Column Temp: 30 C page 28

29 ELSD Application Areas Anyone doing HPLC! Especially those currently using UV, RI, fluorescence, or MS Anyone with non-chromophoric compounds in their sample Anyone requiring more complete peak information (combichem analysis) Anyone requiring nanogram-level or higher detection limits Anyone analyzing non-volatile or semi-volatile samples Anyone trying to collect fractions of non-chromophoric compounds Make sure you see everything in your sample page 29

30 Key Application Areas Pharmaceuticals Non-chromophoric drugs Excipients Impurities Amino acids Aminoglycoside antibiotics Polymers/Surfactants PEGs Polysorbate Tween PVA GPEC Dimethicone Nutraceuticals Ginseng Black Cohosh Ginkgolides Glucosamine Lipids Phospholipids Triglycerides Fatty acids Carbohydrates Simple< sugars Oligomers Sugar alcohols page 30

31 Conclusion Near Universal Detect any compound less volatile than the mobile phase Sensitive Low nanogram detection limits Gradient Compatible Maintain stable baselines during steep gradients Compliment to UV and MS Replacement for RI page 31

32 and Finally.. Any Questions? page 32

Utilizing ELSD and MS as Secondary Detectors for Prep HPLC and Flash Chromatography. Tips and Techniques to Optimize ELSD and MS based Purification

Utilizing ELSD and MS as Secondary Detectors for Prep HPLC and Flash Chromatography Tips and Techniques to Optimize ELSD and MS based Purification Utilizing ELSD and MS as Secondary Detectors for Prep