UV-VIS VIS Detection in HPLC

Transcription

1 3 mv Minutes F ucose 2. Gal actosami ne 3. Gl ucosamine 4. Gal actose 5. Gl ucose 6. Mannose UV-VIS Detection in HPLC UV-VIS VIS Detection in HPLC Shulamit Levin Medtechnica levins@medtechnica.co.il shulal@zahav.net.il The Detector is the Eye of the HPLC System Detector Control & Data Processing Waste a b c d Pump flows 5-5µ L/min) Auto Sampler HPLC Column in Oven Fraction Collector 1

2 Detectors UV/VIS Refractive index Fluorescence Electrochemical Conductivity Mass-spectrometric spectrometric (LC/MS) Evaporative light scattering Optical Bench of UV-VIS VIS Detector Optical Light Path Beam Splitter Mirrors Reference side Sample side Taper-Cell Flow Cell Dual Photodiode Beam-Defining Apparatus Deuterium Arc Lamp Illumination Lens Aperture Slit Rotating Diffraction Grating 19 to 6nm 2

3 Beer's Law Absorbance = Extinction Coefficient x Pathlength x Concentration Reduce Pathlength Reduce Concentration UV Chromophores 3

4 UV Chromophores UV-Vis Vis chromophores λmax Em x λmax Adenine 26.5 E = 13.4 Guanine 275 E = 8.1 Cytosine 267 E = 6.1 Thymine E = 7.9 Uracil E = 8.2 NADH 34 E = 6.23 NAD 26 E = 18 4

5 UV spectrum of 1 nm mobile phase TFA Acetic Acid nm ph nm ph Formic Acid nm ph Sodium Phosphate nm ph 2. U.V. Cut-offs for some Common Solvents Remember Solvents chosen can affect detection!! Solvent UV Cutoff Solvent UV Cutoff Water 18 N-Heptane 197 Methanol 25 Cyclohexane 2 N-Propanol 25 Carbon tetrachloride 265 Acetonitrile 19 Chloroform 245 THF 225 Benzene 28 Acetone 33 Toluene 285 Methyl acetate 26 Methylene chloride 232 Ethyl Acetate 26 Tetrachloroethylene 28 Nitromethane 38 1,2-Dichloroethane 225 All wavelengths reported in nm. 5

6 UV Detection of AccQ-Tag Amino Acid Derivatives Aspartic Acid Glutamic Acid Hydroxyproline Serine Asparagine AMQ Glycine Glutamine Histidine Threonine Arginine Alanine NH3 Proline Alpha-aminobutyric acid Tyrosine Cysteic Acid Vaine Mtehionine Minutes Isoleucine Ornithine Leucine Lysine Phenylalanine Tryptophan Diode Array Detector 6

7 Extraction of 3D Data Chromatogram Absorbance λ1 λ2 Absorbance Spectrum Wavelength Time PDA Spectrum Index Plot DNPH Derivatives.25 ng Each Peak nm Millennium PDA Spectrum Index Plot - SampleWeight.25 ng - PDA 36. nm

8 Coelution of 2 Peaks A Coelution detection at a single wavelength B Coelution is the sum of absorbance of 2 peaks A and B Peak Purity Measurement nm Spectra at apex and inflection points are displayed Spectrum at maximum impurity is different.4.2 Maximum Impurity

9 Maximum Impurity Detection Millennium PDA Spectrum Index Plot - SampleWeight.25 ng 36nm 996PDA 36. nm nm nm.3 Coelution of DNPH.3 Hexaldehyde and.2 2,5-Dimethylbenzaldehyde.2.1 Hexaldehyde 2,5-Dimethylbenzaldehyde Minutes Determination of Peak Purity Peak Purity Spectral Matching S ta n d a r d U n k n o w n Absorbance Time Peak Purity analyzes all spectra (minimum 15) within a peak against the apex spectrum of the peak itself. T im e Spectral match of apex spectrum of the unknown against the apex spectrum of a standard, stored in a user s library. 9

10 Different Spectra 53 deg Ethylparaben EthylPaba Absorbance nm 1 deg of Spectral Contrast Absorbance Theophylline Dyphylline Similar spectra for structurally related compounds nm 1

11 Spectral Contrast.5 Degrees Absorbance Methylparaben Ethylparaben Very similar spectra, CH2 difference Spectral Contrast can differentiate these spectra nm Spectra of non-uv Active Compounds Analyte and 2 Impurities Absorbance nm 11

12 Detectors UV/VIS Refractive index Fluorescence Electrochemical Conductivity Mass-spectrometric spectrometric (LC/MS) Evaporative light scattering Typical LC/MS System Progression SOURCE - - ANALYZER ION DETECTOR DETECTION OF IONS SORTING OF IONS SAMPLE DESOLVATION AND IONIZATION LC/MS INTERFACE HPLC MASS SPECTRUM DATA SYSTEM CHROMATOGRAM 12

13 (1.696) 1: Scan ES e5 % (1.696) 1: Scan ES e5 1 % m/z m/z UV-VIS Detection in HPLC Mass Spectrometer 3D Run UV-Diode Array Chromatogram with poor resolution Mixture Abs Int : Diode Array nm Time Total-Ion-Current MS Chromatogram with poor resolution Mix Mixture Int : Mass Chromatogram Time Selectivity of Mass Spectrometer Detector Mix Mixture Int Time Extracted Ion Chromatogram of a single Component from a mixture of Components Mix : Scan ES e5 % Time 13

14 LC-MS Analysis e4 XTerra MS C18, 2.1 x 5 mm ( 5 µm) 1 N N (4) e4 296 Ding Time (min) e e e5 TIC 3.5e O O N NH OH N H (2) (3) (1) e e4 9.25e Mass/Charge (m/z) 1 µl injection of 2 ng/ml sample (in 4% MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline, 4=Trimipramine, 65/35.1 % Formic Acid / MeCN.2 ml/min Qualification of Detectors Selectivity Sensitivity and detection limit Stability Linear range Dynamic Range Reproducibility Sampling Rate and Response Times Volume Maintenance 14

15 Properties of Detectors SELECTIVITY SPECIFIC UNIVERSAL A selective detector allows one to see only components of interest despite of their co-elution with any others. R E S P O N S E Sensitivity CONCENTRATION Sensitivity of a detector is not the minimum amount that can be detected. 15

16 Signal to Noise h signal = 2 x h noise h signal h noise LOD & LOQ Limit of detection Lowest concentration that can be detected Signal-to-noise ratio of 2:1 or 3:1 Limit of quantitation Lowest concentration that can be determined with acceptable precision. Signal-tonoise ratio of 1:1 16

17 Importance of Sensitivity: Impurity Profile Compound Impurity 1 Retention Time (Minutes) Area %.54 Signal-to- Noise 4.1 Impurity Butamben Impurity Impurity Impurity Detector Noise 17

18 Baseline Stability SHORT RANGE NOISE LONG RANGE DRIFT Noise and Drift Noise, drift, and smallest detectable peak. 18

19 Linearity-Linear Dynamic Range R E S P O N S E Max linear response 2 x detector noise CONCENTRATION Dynamic Range R E S P O N S E Time The range of solute concentration over which the detector continues to respond to changes in solute concentration 19

20 Background Response Background Absorbance Reduces Dynamic Range 2

21 Dynamic Range Effect of Sampling Rate on Peak Shape (UPLC) pt/s 5 pts/s 4 pts/s Minutes 21

22 Time Constant At High Data Rates (UPLC) No Filtering.1s.3s.5s 1.s Minutes Contribution of Volume to Band Broadening Low Volume High VOLUME 22

23 Minutes UV-VIS Detection in HPLC Flow Cell Comparison - Sensitivity.2 Analytic al High Sensitivity Delta Area Height Peak Width at 13.4% Minutes Time constant 23

24 Maintenance Flow Cell & Optics UV-VIS, Fluorescence, RI Electrode Conductivity and Electrochemical Nebulizer ELS and MS Wavelength Accuracy Test 24

25 Specifications 2487 HP11 MWD TSP 3HR Shimadzu 1AP Noise.35x1-5 1x1-5 2x1-5.35x1-5* Drift 1x1-4 2x1-3 2x1-4 2x1-4 Linearity Not Reported** Bandwidth 5nm 1-16nm 2.8nm 8nm Internal Filter Er Ho N/A N/A Lamp D2 D2/W D2/W D2/W * With 2sec TC ** Says Range to