Efficient HPLC Method Development Using Fluorescence Spectral Data

Transcription

1 PittCon 2005 Tanya Jenkins Senior Applications Chemist Jeannine Jordan Product Manager Detection Systems Worldwide Marketing Waters Corporation 34 Maple Street Milford, Massachusetts USA Efficient HPLC Method Development Using Fluorescence Spectral Data

2 Overview Why Choose Fluorescence Detection? Fundamentals of Fluorescence Detection Design of Fluorescence Detectors HPLC Considerations for Fluorescence Challenges of Fluorescence Detection Method Development Advantages of Using Fluorescence Spectral Data

3 Why Fluorescence Detection? Fluorescence detectors (FLDs) are probably the most sensitive among modern HPLC detectors. Typical sensitivity is times greater than that of UV/VIS. Sensitivity typically in pg/fg range however even a single analyte molecule can be detected in the flow cell. Fluorescence detectors are very specific and selective when compared to other optical detectors. Technique is simple and non-destructive

4 Fluorescence S/N = 1258 Why Fluorescence Detection? SENSITIVITY Absorbance S/N = Minutes 70x Better Sensitivity for Anthracene

5 EU EU Why Fluorescence Detection? SELECTIVITY Minutes 240 nm Expanded 230ex/350em 280ex/450em 230ex/310em Licorice Extract Minutes

6 Why Fluorescence Detection? When other detection techniques, such as UV/Vis, are too insensitive or not selective. Environmental PolyAromatic Hydrocarbons Phenols, Carbamates Food and Beverage Aflatoxins Mycotoxins Vitamins (B2, B6) Dyes Biotech and Pharmaceuticals Drugs and their metabolites Derivatized amino acids (AccQ-Tag) or (OPA) When no UV/VIS Chromophores exist. Label with Fluorescence tags

7 Fundamentals of Fluorescence A fluorescent compound absorbs light (UV or Vis) and its molecules reach an excited state. The phenomenon of light emission during this process of returning to the ground state is called fluorescence. Excited State Ground State S 1 S 0 Excitation (1) Vibration Energy (2) Emission (3) (1) Molecules enter an excited state after absorbing UV or visible light. Molecules reach an unstable state of high energy. (2) Electrons lose excess energy as vibration energy and reach the lowest level of excited singlet state. (3) Fluorescence occurs when electrons lose energy and reach the ground state

8 Fundamentals of Fluorescence The most intense fluorescence is found in compounds containing aromatic functional groups with low energy π π* transition levels. Fluorescence can be observed in aliphatic and alicyclic carbonyl structures and highly conjugated double-bond structures. Most unsubstituted aromatic hydrocarbons fluoresce in solution with the quantum efficiency increasing with the number of rings, however simple heterocyclics do not fluoresce Substitution of the rings causes shifts in absorption maxima and efficiency. Fluorescence is favored in rigid structures.

9 Fundamentals of Fluorescence Application Areas H 3 C NH O O O CH 3 O O O O CH 3 Carbamate Pesticides O Aflatoxins O CH 3 H C CH CH 3 CH OH Poly-aromatic Hydrocarbons CH 3 Vitamins H 3 C CH 3 NH 2 OH Amino acids O + ACQ = N H NH O NH CH 3 O CH 3 OH

10 Detector Design Considerations Sensitivity Fluorescence is used over UV/Vis because of high sensitivity, therefore the detector must optimize its light intensity. Noise Performance The second half of sensitivity is minimizing noise. Optics that reduce stray light and minimize scatter will improve noise performance. Low Dispersion Low dispersion is important for the integrity of the separation and preserving the concentration band.

11 Detector Design Considerations Axially Illuminated Flow Cell Design What is it? Excitation energy enters the rectangular flow cell along its long axis allowing the excitation energy to be reflected back along the axis of the cell. λem λex What s the advantage? Provides best S/N specification on the market with lowest noise and minimal RI effects Flow cell axial walls consist of a geometrically matched lens and curved mirror. This gives the light a second opportunity to be absorbed, or exit through the lens, minimizing stray light.

12 Detector Design Considerations Low Dispersion Flow Cell Design What is it? The 2475 excitation and emission optics are at right angles, and also in opposite planes, to minimize stray light 2475 Flow Cell Design Conventional Flow Cell Design λem λem λex λex What s the advantage? A long, thin rectangle disperses liquids less, has less stray light, less volume and more path length than the traditional cubic flow cell. Increase in response, peak areas and heights because path length is longer than conventional cuvette-shaped cells.

13 Detector Design Considerations Use of High Energy Xenon Source What is it? The 2475 Excitation optics uses a curved mirror to focus the most intense part of Xenon emission on to the small flow cell entrance. Area of Most Intense Area of Least Intense Emission Emission I n t e n s i t y Image 2475 Xenon Source Lamp What s the advantage? I n t e n s i t y Conventional Xenon Source Decrease in noise, especially at longer Ex wavelengths Improved wavelength accuracy because of smaller bandwidth Image FLR signal receives the highest quality of excitation light entering the flow cell.

14 Detector Design Considerations More Light into the Flow Cell Relative Lamp Output, Continuous Arc Xenon lamp versus Xenon Flash Lamp Photocurrent, microamps Continuous Arc Xenon Lamp Xenon Flash Lamp Wavelength, nm.

15 Detector Design Considerations Mirrors Not Lenses Mirror Ex Optics - Top View Grating less optical complexity optics are at 90º in 2 different planes. Traditional Optics Xe Lamp Flow cell Flow cell Mirror Em Optics Side View PMT Grating What s the advantage? Stray light is reduced Lenses tend to absorb more light than mirrors and are less efficient mirror based optics optimize signal energy throughput Ex and Em exit can use the same long path length of the rectangular flow cell design

16 Detector Design Considerations Normalized Emission Units What is it? Auto-Optimize Gain uses the Raman peak of water and will determine the recommended Gain setting for an analysis What s the advantage? Normalized EUs eliminate peak dependence on gain settings. Factors that normally influence FLR measurements, such as lamp or optics degradation, can be compensated for. Superior bench-to-bench reproducibility.

17 Detector Design Considerations Normalized Emission Units Naphtalene, emission units (normalised mode) Area count Beta 10 Beta 15 S/N J N Comparison for naphthalene Excitation : 219 nm Emission : 329 nm Area count Naphtalene, energy units Beta 10 Beta 15 S/N J N % RSD with normalization : 18 % % RSD w/o normalization : 60 %

18 HPLC Considerations for Fluorescence Detection Physical characteristics that effect sample fluorescence Quenching effects of solvent (e.g., MeOH vs. ACN). The presence of buffers or ion-pairing reagents in solvent. The concentration of the solvent in sample fraction. The ph of the solvent. The temperature of solvent. The presence of dissolved oxygen in solvent. The concentration of sample being separated. The co-elution of other compounds with the sample. The quality and reproducibility of the HPLC solvent and sample delivery system. Note: All spectra, including UV/VIS, are also influenced by many of the factors listed above. However, these effects are significantly magnified when utilizing fluorescence detection techniques.

19 HPLC Considerations for Fluorescence Detection Comparison of Response Degassed and Undegassed Solvents Degassed Not Degassed Column- Waters PAH 27º C Eluent A: Water Eluent B: Acetonitrile Gradient: 60% B to 100% B using curve 9 in 12 minutes Hold 11 minutes Flow Rate 1.2 ml/min Injection: 20ul 1- Benzo(b)flouranthene- 400 ppb 2- Benzo(k)fluoranthene- 200 ppb 3- Benzo(a)pyrene- 200 ppb

20 HPLC Considerations for Fluorescence Detection Effect of Concentration on Pyrene Excitation Maxima In n-heptane ~ 10 5 M Emission λ ~ 10 3 M Excitation λ From: Turro, N.J., Modern Molecular Photochemistry Menlo Park CA, Benjamin/Cummings Publishing, 1978

21 Method Development Challenges for Fluorescence Detection Each compound has a unique excitation and emission wavelength To get a fluorescence signal you must determine the excitation and emission maxima for each component in the sample and use these exact wavelengths to maximize sensitivity Method development can be very tedious if there are many compounds in your sample One option is to use a bench top spectrophotometer, but what if you don t have pure standards?

22 Method Development Challenges for Fluorescence Detection Naphthalene: Excitation = 245 nm, Emission = 325 nm Energy * Minutes Minutes PAH 610 Standard 16 Components, 15 Fluoresce

23 3D Capabilities Reduce Detection Method Development Time What is it? Allows the user to collect 3- dimensional emission or excitation spectral data on the fly Software designed to process and analyze the data gives the user results faster What s the advantage? 3D data allows for faster methods development Enhanced peak identification through the use of spectral libraries

24 3D Capabilities Reduce Detection Method Development Time Polyaromatic Hydrocarbons Structures

25 3D Capabilities Reduce Detection Method Development Time Polyaromatic Hydrocarbons Method Column: PAH Column 4.6 x 250mm, 5µm Mobile Phase A: Water Mobile Phase B: Acetonitrile Flow Rate: 1.2 ml/min Gradient: 60%-100% B over 12min curve 9, hold 11min Injection Volume: 20.0 µl Sample Diluent: 50/50 Water/ACN ( ng/µL) Needle Wash: 5:1:1 = ACN:Water:IPA Seal Wash: 95/5 Water/ACN Temperature: 27 C FLD Detection: as described Sampling rate: 2 pts/sec Time Constant: 2.0 PMT Gain: 1 Instrument: Alliance 2695/2475 FLD

26 3D Capabilities Reduce Detection Method Development Time Injection #1 - Scan to Determine Optimal Emission λ

27 3D Capabilities Reduce Detection Method Development Time Injection #2 - Scan to Determine Optimal Excitation λ

28 3D Capabilities Reduce Detection Method Development Time Automatically Build a 2D Method

29 3D Capabilities Reduce Detection Method Development Time Injection #3 - Confirm Optimized 2D Method Phenanthrene Energy Naphthalene Anthracene Fluoranthene Pyrene Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(ghi)perylene Acenaphthalene Fluorene Dibenz(ah)anthracene Benzo(a)pyrene Indeno(123-cd)pyrene Minutes

30 3D Capabilities Reduce Detection Method Development Time Why Create a 2D Method from Spectral Data? SENSITIVITY Base sampling rate of detector is a set value With lower sampling rates, points are averaged to provide the required sampling rate Averaging of data points in 2D Mode at lower data rates results in better baseline noise

31 3D Capabilities Reduce Detection Method Development Time 0.30 Noise Comparison between 2D and 3D Mode 0.20 Energy D Mode 1 pt/s Minutes Energy D Mode 1 pt/s Minutes

32 Library Matching Capabilities Another Benefit of 3D Spectral Information Spectra of samples/standards can be collected and stored in a library for comparison. Spectral contrast theory measures the differences between the spectra in the library and the spectrum collected for the unknown peak. The probability that the two spectra match depend on the degree of difference between the spectra as compared to spectral differences due to non-ideal behavior such as noise, linearity, and solvent effects

33 Library Matching Capabilities Spectral Contrast Theory Each compound has a unique spectrum that is represented by a vector in space. Spectral Contrast Angle is the angle between vectors, the differences. A value of zero degrees the vectors overlay and suggest that the two spectra are equivalent. A value of 90 degrees demonstrates maximum differences in the two spectra

34 Library Matching Capabilities Spectral Contrast Theory Absorbance θ Spectrum A Noise Spectrum B Noise Angle is the gray area, the area of uncertainty The vector length is proportional to absorbance Detection limits for impurities and ability to identify very small peaks are directly link to the noise. A good detector must provide at the same time both high sensitivity and high resolution

35 Library Matching Capabilities Spectral Contrast Theory Spectral Contrast 53 Degrees Absorbance Ethylparaben Ethyl-PABA 53 degrees is a large spectral difference Wavelength (nm)

36 Library Matching Capabilities Spectral Contrast Theory Spectral Contrast 10 Degrees Absorbance Theophylline Dyphylline Similar spectra for structurally related compounds Wavelength (nm)

37 Library Matching Capabilities Spectral Contrast Theory Spectral Contrast 0.5 Degrees Methylparaben Ethylparaben Very similar spectra, CH2 difference Absorbance Spectral Contrast can differentiate these spectra Wavelength (nm)

38 Library Matching Capabilities Fluoroquinolone Method Column: Atlantis Column 4.6 x 150mm, 3µm Mobile Phase A: 0.2% HFPA in Water Mobile Phase B: Acetonitrile Mobile Phase C: Methanol Flow Rate: 1.1 ml/min Isocratic: 75:22:3 A:B:C Injection Volume: 10.0 µl Sample Diluent: 0.1ng/µL in Water Needle Wash: 5:1:1 = ACN:Water:IPA Seal Wash: 95/5 Water/ACN Temperature: 30 C FLD Detection: ex280nm, em nm, extracted 460nm Sampling rate: 1 pts/sec Time Constant: 1.0 PMT Gain: 100 Instrument: Alliance 2695/2475 FLD C H 3 HN N H 3 C N N F Enrofloxacin N F Ofloxacin N F O N O N O CH 3 Norfloxacin CH 3 N HN COOH H 3 C COOH HN COO H N F N F F N N O O COOH Ciprofloxacin CH 3 COOH Lomefloxacin

39 Library Matching Capabilities Separation of 5 Fluorquinolones EU Minutes ex280nm, em nm, extracted 460nm

40 Library Matching Capabilities Emission Data for all 5 Fluoroquinolones

41 Library Matching Capabilities Sample: Enrofloxacin

42 Library Matching Capabilities Library Matching adds Increased Confidence in Peak Identification EU Norfloxacin Ofloxacin Ciprofloxacin Lomefloxacin Enrofloxacin Minutes

43 Library Matching Capabilities Vitamin Analysis Mobile Phase A: Isopropanol Mobile Phase B: Hexane Flow Rate: 2.0 ml/min Dial-a-Mix: 1% A, 99% B Injection Volume: 5.0 µl Sample Diluent: Hexane Needle Wash: IPA Seal Wash: IPA Temperature: 25 C FLD Detection: ex. 295, em. 325 Sampling rate: 1 pts/sec Time Constant: 2.0 PMT Gain: 1 Instrument: Alliance 2695/2475 3D FLD Vitamin A Vitamin E

44 Library Matching Capabilities Vitamin Separation Ex λ 295nm; Em λ nm, extracted 325

45 Library Matching Capabilities Overlay of Emission Spectra for Vitamin A and E

46 Library Matching Capabilities Overlay of Vitamin E Emission Spectra with 2 Closest Library Matches

47 Library Matching Capabilities Overlay of Excitation Spectra for Vitamin A and E

48 Library Matching Capabilities Overlay of Vitamin A Emission Spectra with 2 Closest Library Matches

49 Peak Purity with Fluorescence Spectral Data Peak purity algorithms compares the spectrum collected at the apex of a peak to the other spectra collected across the peak. For a peak purity test to be successful, the spectra of the co-eluters needs to be notably different. Fluorescence detectors have large bandwidths to increase sensitivity which decreases the spectral definition.

50 Peak Purity with Fluorescence Spectral Data Good optical resolution gives good quality spectral information Benzene spectra maxima spaced 2.5nm Less resolution at 3.6 nm vs. 1.2 nm nm UV maxima shifted

51 Peak Purity with Fluorescence Spectral Data A A θ B θ Larger spectral differences B More of B relative to A θ A B B A B not detectable Detection of coelution B when analyzing for A Peak Purity should not be used with Fluorescence Data

52 Summary Fluorescence is used for detection when increased sensitivity or selectivity is needed Method development for fluorescent methods can be much more difficult because the excitation and emission maxima must be determined 3D scanning capabilities can help to drastically reduce detection method development time by allowing for excitation and emission spectra to be collected to determine optimal wavelengths Library matching is a powerful tool which helps with compound identification

53 The Fluorescence Method Development Solution Waters 2475 Multi λ Fluorescence Detector Performance by Design for Efficient Method Development

54 Appendix 1 Separation Conditions for Anthracene Chromatographic Conditions: Column: Atlantis 4.6 x 150mm, 3µm Mobile Phase A: Water Mobile Phase B: Acetonitrile Flow Rate: 1.0 ml/min Isocratic: 30/70 Water/ACN Injection Volume: 2.0 µl Sample Diluent: 50/50 Water/ACN (100ng/µL) Needle Wash: 5:1:1 = ACN:Water:IPA Seal Wash: 95/5 Water/ACN Temperature: 30 C PDA Detection: nm, extracted 249nm Time Constant: 0.5 FLD Detection: ex249, em402 Sampling rate: 5 pts/sec Time Constant: 0.5 PMT Gain: 10 Instrument: Alliance 2695/2475 3D FLD/2996 PDA

55 Appendix 2 Separation Conditions for Licorice Extract Chromatographic Conditions: Column: Atlantis 4.6 x 150mm, 3µm Mobile Phase A: Water Mobile Phase B: Acetonitrile Flow Rate: 1.0 ml/min Gradient: 10%-98% B over 20min, hold 10min Injection Volume: 10.0 µl Needle Wash: 5:1:1 = ACN:Water:IPA Seal Wash: 95/5 Water/ACN Temperature: 40 C PDA Detection: nm Time Constant: 1.0 FLD Detection: ex230nm, em nm Sampling rate: 1 pts/sec Time Constant: 0.5 PMT Gain: 1 Instrument: Alliance 2695/2475 3D FLD/2996 PDA