Introduction to UV-based detectors

UV detectors overview

1. Jan Pettersson Nordic HPLC & Chromeleon CDS Support Specialist Thermo Fisher Scientific Introduction to UV-based Detection

2. UV Vis Detectors The ideal detector ? Why do we get a signal? Optics Lamps Flow cell Band and slit width Data collection rate and time constant Reference Stray light , refractive index effects & noise Thermo Scientific™ Vanquish ™ HPLC

3. UV Vis Detectors – The ideal detector? A workhorse for detection and quantitation of organic compounds Fast , accurate and sensitive Quick and easy to set up and require only basic maintenance However – to optimize performance one needs to understand the basic physics of the absorption process and the working principles of the various instrument designs This will lead to improved sensitivity, robustness and quantitation. MWD DAD VWD

4. UV Vis Detectors The ideal detector ? Why do we get a signal ? Optics Lamps Flow cell Band and slit width Data collection rate and time constant Reference Stray light , refractive index effects & noise

5. Why Do We Get a Signal? V ery simplified: The light from the lamp excites the electrons in the sample to a higher state of energy. Different molecules absorbs light at different frequencies. The shorter the wavelength the higher the energy

6. Structures with double bonds often absorb UV light due to the presence of p electrons Hetero atoms having non-bonding valence-shell electron pairs also absorb UV light Conjugated systems are particularly useful in UV spectrometry Why do we get a signal?

7. Above 200 nm excitation of electrons from p-, d-, and π-orbitals and especially conjugated systems produce informative spectra UV Chromofores

8. UV spectra often contain broad responses due to vibrational and rotational transitions which accompany electronic transitions of bonding electrons

9. Series of alternating single and double or triple bonds represent a conjugated system Addition of double bonds increases the wavelength by around 30nm Intensity of absorption increases due to delocalized p electron systems giving rise to a larger number of ‘pseudo’ double bonds Conjugation effects

10. Solvent Effects – Polarity UV-Visible spectrum of propanone in hexane UV-Visible spectrum of propanone in water

11. Solvent Effects – Why W ater and ACN are Popular Solvent (nm) Minimum wavelength Acetonitrile 190 Water 191 Cyclohexane 195 Hexane 201 Methanol 203 Ethanol 204 Ethoxyethane 215 Dichloromethane 220 Trichloromethane 237 Tetrachloromethane 257

12. pH Effects Anthocyanin pigment in buffers of varying pH

13. pH Effects Anthocyanin pigment in buffers of varying pH

14. Expansion of the solvent, may be sufficient to change the apparent absorbance and thereby the accuracy of quantitative results Temperature may affect equilibria, which can be either chemical or physical (denaturation of nucleic acids for example) For organic solvents, changes in refractive index with temperature can be significant Convection currents cause different temperatures to occur in different parts of the cell, the resulting Schlieren effect can change the apparent absorbance. To control this problems have as stable temperature as possible! Temperature effects

15. UV Vis Detectors The Ideal detector ? Why do we get a signal ? Optics Lamps Flow cell Band and slit width Data collection rate and time constant Reference Stray light , refractive index effects & noise

16. Two UV/Vis detectors Forward optics design => variable wavelength detector (VWD) => no real-time spectra information available Reversed optics design => diode array detector (DAD) => includes real-time spectra information Operating Principle – Optics Design

17. Operating Principle: Variable Wavelength Detector (VWD) Forward optics d esign Only the selected wavelength passes the flow cell A part of the light beam is redirected to the reference diode Light source  Dispersion device  Flow cell  Sample diode

18. Operating Principle: Wavelength Diode Array Detctor (DAD) Reversed optics d esign Light beam passes the flow cell before being diffracted No true reference signal can be obtained Any diode or bunch of diodes can be selected as a reference Light source  Flow cell  Dispersion d evice  Diode array

19. Uses of Diode Array Detectors Peak purity measurement Signal deconvolution / Multiple wavelength acquisition Dynamic spectral acquisition and identification 254 nm 220 nm

20. Operating Principle – 2 nd Order Filter Light diffraction at a dispersion device results always in different orders of light segmentation No filter Dispersion device (grating or prism) ‘White’ light 0-order 1st-order 2nd-order

21. Source / UV Lamp Deuterium lamp – silica quartz envelope to reduce noise and transmit at lower wavelengths mounted on a pre-set (focused) mount Need a stable source with adequate intensity over a suitable wavelength range Over time the intensity decreases – 1000h half life is typical It loose about 6 hours every time it is switched on and off

22. UV Vis Detectors The Ideal detector ? Why do we get a signal? Optics Lamps Flow Cell Band and Slit Widht Data Collection Rate and Time Constant Reference Stray light,Refractive Index Effects & Noise

23. Source / Vis Lamp Tungsten Halogen lamp for the longer wavelengths Light from both sources can be mixed to generate a single broad band source

24. ‘ UVLampIntensity ‘: measured at 254 nm The reading is normally above 3 million counts (narrow slit width) for the UV lamp for an analytical SST cell, and should be above 1 million counts at least ‘ VisLampIntensity ‘: Expected value: above 2 million counts Beside intensity value noise level and signal-to-noise ratio need to be checked (application depending) Guidance values: VWD: OQ limit 50%, PQ Limit 40% DAD: > 3.000.000 counts (depending on slit width, here: narrow) Lamp behavior Intensity high at the beginning of lifetime unstable and highly drift and noise affected Significant intensity drop within the first ~ 100 hours afterwards slowed and steady decrease Source / Vis Lamp

25. UV Vis Detectors The Ideal detector ? Why do we get a signal? Optics Lamps Flow Cell Band and Slit Widht Data Collection Rate and Time Constant Reference Stray light,Refractive Index Effects & Noise

26. Flow Cell – Signal to Noise Signal height Light path should be as long as possible Small flow cell volume, small inner diameter measurement channel Sample Lamp Detector Flow in Flow out

27. Flow Cell – Signal to Noise Signal height Light path should be as long as possible Small flow cell volume, small inner diameter measurement channel Sample Lamp Detector Flow in Flow out To reduce Noise Get a s much light as possible through flow cell 4 x light > 0.5 x noise

28. Flow Cell Volume Flow cell volume should not exceed 10% of the peak volume Flow cell ok Peak Volume Cell volume Flow Flow cell too big Micro column peak volumes Flow cell ok Micro column peak volumes Smaller cell volume  less light is passing through the flow cell

29. Flow Cells – Thermo Scientific™ UltiMate ™ 3000 HPLC System

30. Inlet Outlet Light Flow path Flow cell locks Glass body Flow cell Fluidics Fiber optics Vanquish Light Pipe Flow Cells – Fused Silica

31. Overlay of chromatograms obtained from different test setups VDAD: higher (by 30-50%) and narrower peaks (by 30-35%) Column: Thermo Scientific Hypersil Gold, C18, 2.1 ×100 mm, 1.9 µm, P/N 25002-102130 System: Binary UltiMate 3000 Mixer Vol.: 200 µL Mobile Phase: A – Water B – ACN Flow rate: 0.7 m L/min Pressure: 630 bar (max) Temperature : 35 ºC Injection: 1 µL Detection: DAD-3000RS, semi-micro or analytical flow cell, wide slit (4 nm) Vanquish DAD with standard flow cell, 4 nm slit 254 nm, 4 nm bandwidth, 20 Hz, 0.2 s response time Analytes: 1. Uracil 2. Acetanilide 3. – 10. Homologous Phenones 50 µg/mL each 0.00 3.50 -50 500 ACN: 40 % 100 % 40 % 1 2 3 4 5 6 7 8 9 10 min mAU 0.30 0.40 -30 250 1 min mAU Vanquish DAD, 2 µl standard flow cell DAD-3000RS, 2,5 µl semi-micro flow cell DAD-3000RS, 13 µl analytical flow cell Binary UHPLC Gradient Performance

32. VWD 11µl 10mm Standard Analytical flow cell 2,5µl 5mm Semi-Micro flow cell 45nl 10mm Capillary flow cell 3nl 10mm Nano flow cell Flow Cells DAD 13µl 10mm Standard Analytical flow cell 5µl 7mm Semi-Analytical flow cell 2,5µl 5mm Semi-Micro flow cell Vanquish 2µl 10mm Standard Analytical Light Pipe flow cell 13µl 60mm Light Pipe flow cell

33. UV Vis Detectors The Ideal detector ? Why do we get a signal? Optics Lamps Flow Cell Band and Slit Widht Data Collection Rate and Time Constant Reference Stray light,Refractive Index Effects & Noise

34. Operating Principle – Slit Width VWD detector Bandwidth is defined by entrance slit At 254nm the bandwidth is 6nm (254nm +/- 3nm)

35. Diode Array Detector Slit width Slit defines optical resolution and therefore minimal physically meaningful bandwidth Flow Cell

36. Diode Array Detector Bandwidth S/N ratio Spectral resolution ↑ ↑ ↓ ↓ ↓ ↑ Slit w idth Baseline noise Spectral resolution ↓ ↑ ↑ ↑ ↓ ↓ Slit width Bandwidth Flow Cell Slit defines optical resolution and therefore minimal physically meaningful bandwidth

37. Effects of Slit Width Slit width - 1nm 4 x light > 0.5 x noise

38. Effects of Slit Width Slit width - 16nm 4 x light > 0.5 x noise

39. Setting Bandwidth Bandwidth 4 nm s/n = 5 4 x light = < 0.5 noise

40. Setting Bandwidth Bandwidth 4 nm s/n = 5 Bandwidth 30 nm s/n = 25 4 x light = < 0.5 noise

41. Linearity of butylparabene Measured at 240 nm The linearity is excellent between 1-4 nm bandwidth From 8 -100 nm the linearity permanently decreases 190 250 300 350 402 -10.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 194.1 257.6 nm % Butylparabene Influence on Linearity – Bandwidth

42. Linearity of butylparabene Narrow slit always provides both a slightly better correlation coefficient and a slightly better linearity RSD Influence on Linearity –Slit width

43. Recommended Parameters: Data Acquisition 250 250 0,100 0,105 0,110 0,115 0,120 50 100 150 200 Absorbance [ mAU ] t [min] 0,100 0,105 0,110 0,115 0,120 50 100 150 200 Data rate: 5 Hz t [min] Absorbance [mAU] Data rate: 100 Hz Too few data points effect peak form, reproducibility and area precision A minimum of 20, ideally 30-40 data points/peak is required

44. Data Collection Rate 50Hz 2Hz

45. Time Constant The r ise t ime (Response time ) is closely releated to the time c onstant:Rise t ime = 2,2 x Time constant

46. Data Collection Rate and Time Constant Noise is much more influenced by time c onstant than by data collection rate TC 2.0 s TC 0.2 s TC 0.06 s TC 0.03 s TC 0.01 s DCR 1 Hz DCR 10 Hz DCR 25 Hz DCR 50 Hz DCR 100 Hz

47. Sampling and Rise Time The same instrument, back pressure loop, eluent and sample. The area is the same – the peakshape is very different. 2,5 Hz 2s response time 10 Hz 0,5s response time

48. Automated Settings The Program Wizard of Thermo Scientific Chromeleon™ 7.2 CDS has a dedicated step for setting the correct ‘Data Collection Rate’ and ‘Time Constant’ The internal calculation is based on the peak width at half peak height of the slimmest peak in the chromatogram

49. UV Vis Detectors The Ideal detector ? Why do we get a signal? Optics Lamps Flow Cell Band and Slit Widht Data Collection Rate and Time Constant Reference Stray light,Refractive Index Effects & Noise

50. Operating Principle – Reference on a DAD Light beam passes the flow cell before diffracted No true reference signal can be obtained Any diode or bunch diodes can be selected as a reference If selected reference and acquisition wavelength are the same, the resulting signal will be zero (0) Select a reference wavelength in a ‘quiet’ area of the spectrum (where little absorbance occurs) Reference wavelength range should not interfere with absorbance range of any compound of interest

51. Operating Principle – Reference on a DAD A reference can compensate for Fluctuations in lamp intensity Changes in absorbance/refractive index during gradient analysis Background noise

52. Issues with Reference Wavelength Blue Chromatogram: Without reference Black Chromatogram: With reference Wavelength : 254 nm Both the UV and Vis lamps turned on R eference wavelength set to 600 nm (80 nm bandwidth)

53. UV Vis Detectors The Ideal detector ? Why do we get a signal? Optics Lamps Flow Cell Band and Slit Widht Data Collection Rate and Time Constant Reference Stray light,Refractive Index Effects & Noise

54. Stray Light Stray light is radiation emerging from the monochromator of all wavelengths other than the bandwidth at the selected wavelength Arise from imperfections in the grating, optical surfaces, diffraction effects as well as wider bandwidth and slit width settings   ε = Molar absorption coefficient (dm 3 mol -1 cm -1 ) l = Path length (cm) c = Concentration ( mol dm -3 )

55. Stray Light 0.00% Stray light 0.01% Stray light 0.10 % Stray light 1.00 % Stray light True absorbance (AU] Measured absorbance [AU] Primary effect is to reduce analyte absorbance / Sensitivity

56. Noise: Definition Short term (Statistical) signal changes Defined in ASTM Defines LOD and detection accuracy

57. Noise Effects measurements on analytes with low absorbance Also affects high absorbance samples

58. Noise Usable linear range 0.1 – 1 AU Stray light error Stray light + noise error curve Stray light –noise error curve % Error Absorbance (AU]

59. Webinar: Introduction to UV-based Detection Thank You! Any Questions?