Important_Notices_for_Daily_Use_of_HPLC.ppt
- 1. Important Notices for Daily Use of HPLC Analytical Applications Department Shimadzu Corporation
- 2. 2 Important notice for Mobile Phases Important notice for Sample Solvents Important notice for Column Temperature Important notice for Detection Contents
- 3. 3 Important Notice for Mobile Phases
- 4. 4 Water contains a lot of other things, including: - Organic and inorganic substances - Ionic and nonionic substances - UV adsorbent and fluorescent substances Selecting Water (1) - Lab-made … distilled water, ion-exchange water, ultrapure water, etc. (Ultrapure water: treated by reverse osmosis, ion-exchange membrane, membrane filter, UV irradiation, etc.) - Commercial … distilled water, ion-exchange water, purified water, pure water, HPLC-grade distilled water, etc.
- 5. 5 Example peaks in a blank gradient with no sample injection Selecting Water (2) Peaks of impurities in water Water impurities retained in the column are eluted by acetonitrile. 100% acetonitrile 100% water
- 6. 6 What sort of water should we select? Distilled water, ion-exchange water, purified water, pure water, ultrapure water … Selecting Water (3) HPLC-grade distilled water … reduces absorbance in short UV wavelength region Gradient elution method for reversed-phase HPLC Less baseline drift and ghost peaks Ultrapure water … reduces all impurities General HPLC, ion chromatography Safe to use for many purposes
- 7. 7 Clean water is easier to be contaminated Selecting Water (4) Restrict the causes of contamination Examples: work environment, equipment, handling methods, etc. Equipment producing ultrapure water needs strict maintenance Examples: reverse osmosis membrane, ion-exchange cartridges, etc. Take care with storage containers Examples: leaching of UV-absorption agents from polyethylene containers
- 8. 8 Selecting Organic Solvent (1) Acetonitrile and methanol …absorbance HPLC-grade acetonitrile has low absorbance Methanol Acetonitrile Low absorbance in short wavelength region HPLC-grade acetonitrile is good for analysis in the short (UV) wavelength range. Special grade Special grade HPLC grade Wavelength (nm) Wavelength (nm) Absorbance (AU) Absorbance (AU)
- 9. 9 Acetonitrile has - Low viscosity Lower column pressure load at same flow rate - Small viscosity change when mixed with water Small column pressure changes during gradient analysis It is soft to the column. Acetonitrile and methanol … viscosity Selecting Organic Solvent (2) Water/methanol Water/methanol Water/acetonitrile Water/acetonitrile Pressure (MPa) Water (100%) Organic solvent (100%) Water/Organic solvent 1:1
- 10. 10 Acetonitrile and methanol … elution However, the elution power relationship may reverse with high proportions of organic solvent. (right diagram) Characteristics of a protic solvent (methanol) and an aprotic solvent (acetonitrile) Generally, acetonitrile has higher elution power Methanol25/water75 Acetonitrile10/water90 Methanol95/water 5 Acetonitrile95/water 5 試料:コレステロール 試料:カフェイン Selecting Organic Solvent (3) Sample: caffeine Sample: cholesterol
- 11. 11 Acetonitrile is safe to use for most analyses. Use methanol for poor selectivity or peak shape. Try methanol for samples with poor solubility in acetonitrile. Selecting Organic Solvent (4) Acetonitrile and methanol … Which one to select?
- 12. 12 • Water/Methanol = 1/1 (v/v) – Measure 500 mL water and 500 mL methanol separately and mix them together. – Total does not make 1 L. • 50% (v/v) Methanol water solution – Put 500 mL methanol in a 1L volumetric flask and add water to make 1 L. – Higher proportion of water 1L Water 500 mL Methanol 500 mL Methanol 500 mL Water Water/organic solvent mixing methods and notation Mobile Phase Preparation (1)
- 13. 13 Different results from different mixing methods 1 2 3 3 1 A B 2 Peaks 1: Acetaminophen 2: Dihydrocodeine 3: Caffeine 0 2 4 6 8 10 min Mobile phase A: 20 mmol/L Sodium(phosphate) buffer <pH 2.5>/Acetonitrile = 9:1 (v/v) Mobile phase B: 20 mmol/L Sodium(phospate) buffer <pH 2.5> containing 10% (v/v) acetonitrile Mobile Phase Preparation (2)
- 14. 14 How do we make 20 mmol/L sodium phosphate buffer (pH2.5)? A: If “20 mmol/L” is considered as the phosphate concentration. Mix 10 mmol phosphoric acid and 10 mmol sodium dihydrogenphosphate and dissolve in 1 L of water. B: If “20 mmol/L” is considered as the sodium concentration. Dissolve 20 mmol sodium dihydrogenphosphate in 1 L of water and then drop phosphoric acid to adjust pH to 2.5. C: If “20 mmol/L” is considered as the phosphate and sodium concentration pH is adjusted with perchloric acid. Dissolve 20 mmol sodium dihydrogenphosphate in 1 L of water and then drop perchloric acid to adjust pH to 2.5. Preparation of buffer solution and notation Mobile Phase Preparation (3)
- 15. 15 Different results from different buffer preparation methods Mobile phase A: considered as 20 mmol/L phosphoric acid Mobile phase B: considered as 20 mmol/L sodium Mobile phase C: considered as 20 mmol/L phosphoric acid and 20 mmol/L sodium min 1 2 3 3 1 A B Peaks 1: Acetaminophen 2: Dihydrocodeine 3: Caffeine 2 4 6 8 10 C 1+2 3 2 0 Mobile Phase Preparation (4)
- 16. 16 1)100 mmol/L (sodium) phosphate buffer (pH2.1) - Sodium dihydrogenphosphate dihydrate (MW=156.01) 50 mmol (7.8 g) - Phosphoric acid (85%, 14.7 mol/L) 50 mmol (3.4mL) - Dissolve in water to make 1L. 2)10 mmol/L (sodium) phosphate buffer (pH2.6) - Sodium dihydrogenphosphate dihydrate (MW=156.01) 5 mmol (0.78 g) - Phosphoric acid (85%, 14.7 mol/L) 5 mmol (0.34 mL) - Dissolve in water to make 1L. - Alternatively, dilute 1) 10 times. Preparation of phosphoric acid buffer without using a pH meter Mobile Phase Preparation (5)
- 17. 17 If mobile phase buffer capacity is weak… Peak shapes of acids and bases with pKa near mobile phase pH deteriorate. Column : STR ODS-II (150mmL. × 4.6mm I.D.) Mobile : 20mM buffer solution (pH 4.5) phase / acetonitrile (3/1, v/v) Flow rate : 1mL/min Temp. : 40ºC Detection: UV-VIS (240nm) Left figure: citric acid buffer solution (pH 4.5) used as mobile phase Right figure: phosphoric acid buffer solution (pH 4.5) used as mobile phase pH Buffer Capacity and Peak Shape
- 18. 18 Changes in absorbance dependent on the solvent Changes in absorbance due to dissolved air Effects of Dissolved Air (1) Methanol THF Hexane Acetonitrile Water Spectra of Increased Absorbance Due to Air Saturation
- 19. 19 Column: ODS Mobile phase: Methanol/water (85/15, v/v) Caused by differences in dissolved air volume in the mobile phase and sample solution due to degassing the mobile phase. Appearance of peaks due to dissolved air With mobile phase degassing Without mobile phase degassing Flowrate: 1.0 mL/min Detection: UV 210 nm Effects of Dissolved Air (2) O2 bubbling Air saturation He bubbling O2 bubbling Air saturation He bubbling
- 20. 20 Confirming peaks due to dissolved air Effects of Dissolved Air (3) Mobile phase Pump Injector Column oven Column Detector Degasser Air mixed in Degassed Sample solution Air saturation in mobile phase and sample solution Check by injecting degassed effluent Dissolved air is injected
- 21. 21 Measures to deal with peaks due to dissolved air Effects of Dissolved Air (4) Change the type of organic solvent If methanol is used, change to acetonitrile that has little change in absorbance due to dissolved air. Change the mobile phase composition Lower the organic solvent ratio (particularly with methanol). Change the column Use a column with a smaller retention capacity.
- 22. 22 min 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 mAU 0 100 200 300 400 BPA << With degassing << Without degassing Analysis of Bisphenol A by Fluorescence Detection Quenching phenomenon in fluorescence detection Degassing results in increased fluorescence intensity Effects of Dissolved Air (5) Quenching occurs due to dissolved oxygen
- 23. 23 Important Notice for Sample Solvents
- 24. 24 Peak shape changes due to solvent composition Peak shape deteriorates as injection volume increases Appropriate choice of sample solvent? Sample solvent composition and injection volume Problems due to Sample Solvents
- 25. 25 Change in peak shape due to sample solvent Left: Sample solvent = water/acetonitrile (3/1, v/v) Right: Sample solvent = acetonitrile Column : STR ODS-II (150mm L. x 4.6mm i.d.) Mobile phase: 20 mmol/L citric acid-sodium buffer (pH 4.5)/Acetonitrile (3/1, v/v) Flowrate : 1.0 mL/min Temperature : 40ºC Detection : UV240 nm Peaks spread! Sample Solvents and Peak Shape
- 26. 26 Reversed phase with water/methanol mixture as mobile phase Mobile phase flow Methanol solvent Sample components Mobile phase flow Water solvent Sample components Behavior of sample solvent and solute in the column Analyte band spreads out in methanol, which has large elution power Sample Solvents and Analyte Band
- 27. 27 Difference in elution power affects the theoretical plate number High injection volumes possible with low-elution- power sample solvent Take care if the sample solvent has higher elution power than the mobile phase. Sample Solvents and Injection Volume (1) Sample solvent: water Sample solvent: methanol /water = 3/7 (same as mobile phase) Sample solvent: methanol HPLC conditions Sample: caffeine 10 ug Column: Shim-pack CLC-ODS (150 mmL x 6 mm ID) Mobile phase: methanol/water=3/7 Flow rate: 1 mL/min Temp: room temp. Detection: 270 nm Theoretical plate number Injection volume uL
- 28. 28 Peaks deform due to injection volume Peak shape can be improved by reducing the injection volume Sample Solvents and Injection Volume (2) Elution starts from around 3 min. when 100 uL is injected. HPLC conditions Same as previous slide Analyte caffeine Sample solvent: methanol
- 29. 29 Adsorption of basic substances onto glass surface Beware of losses due to adsorption onto vial Sample solvent Aqueous solution of perchloric acid Water No. of analy sis caffeine thiamine caffeine thiamine 1st 50248 52974 50327 48228 2nd 50120 52691 50372 48293 3rd 50268 53031 50479 48434 4th 50320 52801 50259 48060 5th 50443 52722 50224 47951 ave 50280 52844 50332 48193 RSD(%) 0.23 0.29 0.20 0.40 Sample Solvents and Adsorption Sample solvents : a) aqueous solution of perchloric acid, b) water Sample concentration : caffeine, thiamine both 10 mol/L Injection volume : 10 L
- 30. 30 Important Notice for Column Temperature
- 31. 31 Analysis at constant temperature Enhanced accuracy (repeatability, reproducibility) Effects of Column Temperature Control Enhanced column efficiency Decreased column pressure Enhanced sample solubility, etc. Analysis at higher temperature Analysis at lower temperature Enhanced selectivity (in particular case) Enhanced sample stability, etc.
- 32. 32 Different behaviors of components Column Temperature and Selectivity Peaks 1. Benzoic Acid 2. Sorbic Acid 3. Methylparaben 30C 25C 20C Sometimes separation can be controlled without changing the mobile phase composition.
- 33. 33 Anomer separation in the column Separation of sugar anomers by the ligand-exchange method Column Temperature and Peak Shape (1) Peaks 1. Sucrose 2. Glucose 3. Sorbitol Column : SCR-101C (Ca type) Mobile phase : water Flowrate : 0.8 mL/min Detection : differential refractive index Increasing the column temperature and increasing the anomer equilibration speed forms a single peak. Room temp. (25C)
- 34. 34 Temperature gradient in the mobile phase in the column Heating creates a broad peak. Effect increases at larger column I.D. and mobile phase flow rate <Countermeasure> Connect a preheat coil before the column inlet or auto-sampler. Analyte band spreads out Temperature gradient in the column Column Temperature and Peak Shape (2)
- 35. 35 0 0.5 1 1.5 2 2.5 min 0 0.5 1 1.5 2 2.5 min Without pre-heat coil Column : ODS (50mmL. x 3 mm ID) Mobile phase : water/acetonitrile (3/2, v/v) Flowrate : 1.0 mL/min Effects of the pre-heat coil With pre-heat coil Temperature : 50C Detection : UV 260 nm Sample : parabens Column Temperature and Peak Shape (3)
- 36. 36 Important Notice for Detection
- 37. 37 Effects of instrument environment Detection and Temperature In a room with high temperature fluctuations or near an airconditioner outlet Generally, refractive index detectors, conductivity detectors, and electrochemical detectors are sensitive to temperature effects, causing baseline drift. But UV detectors and fluorescence detectors are also affected by temperature fluctuations.
- 38. 38 - Fluorescence intensity decreases as the temperature rises. - Fluorescence intensity fluctuates due to temperature fluctuations. As the temperature increases, intermolecular collisions increase, resulting in losses in potential energy Fluorescence Intensity and Detection Temperature Maintain the room temperature as constant as possible. >> Enhanced repeatability Set the detection temperature as low as possible. >> Enhanced sensitivity Temperature in Fluorescence Detection (1)
- 39. 39 Room-temperature fluctuations and detector cell inlet temperature Temperature in Fluorescence Detection (2) Changes in cell inlet temperature due to room temperature fluctuations (at 1 mL/min. flow rate) Time (minutes) Temperature (C) Room temperature Liquid temperature (no temp. control) Liquid temperature (with temp. control)
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- 168. 168 Effects of room-temperature fluctuations and cell temperature control Area value decreases 13.5％ from 20°C to 25°C Without cell temp. control With cell temp. control Component: acridine At 20 At 25°C At 20°C, 25°C Time (minutes) Area value decreases 2.2％ from 20°C to 25°C Time (minutes)
- 169. 169 Effects of room temperature fluctuations on mobile phase absorbance Baseline stabilization due to cell temperature control of UV detector Temperature for UV Detection (1) Mobile phase: phosphoric acid–sodium buffer solution (pH 7), detection :UV 210 nm Without cell temperature control Cell temp. Cell temp. Room temp. Baseline Room temp. Baseline With cell temperature control
- 170. 170 Enhanced precision due to cell temperature control 0 5 10 min 0 5 10 min Without cell temp. control With cell temp. control Column : ODS (150 mm x 4.6 mm ID) Mobile phase : phosphoric acid buffer (pH 7)/Methanol (3/1, v/v) Flow rate : 1.0 mL/min Temperature : 40C Detection : UV-VIS detector with temperature control (210 nm) Sample : caffeine (1 ng) Without cell temp. control With cell temp. control 1st 1.000 1.000 2nd 0.987 0.999 3rd 0.963 1.008 4th 0.992 0.994 5th 1.031 0.995 6th 1.026 1.008 RSD (%) 2.55 0.61 Temperature for UV Detection (2)
- 171. 171 Detector Response (1) 0 0.5 1 1.5 min 0 0.5 1 1.5 min 0 0.5 1 1.5 min Response: 50 ms Theoretical plate number: 2642 (Peak 4) Noise: 21.1 AU Response: 500 ms Theoretical plate number: 2426 (Peak 4) Noise: 5.2 AU Response: 1500 ms Theoretical plate number: 1400 (Peak 4) Noise: 0.5 AU Column : ODS (50 mmL. x 3 mm ID) Mobile phase : Water/acetonitrile (1/1, v/v) Flow rate : 1.0 mL/min Peak shape changes due to response Temperature : 25°C Detection : UV260 nm Sample : parabens
- 172. 172 Response setting according to peak width Detector Response (2) Response Suitable peak width 50 ms Unlimited 100 ms 1s or more 500 ms 4s or more 1000 ms 7s or more 1500 ms 10s or more 0.8 1 1.2 1.4 1.6 1.8 2 2.2 3 4 5 6 7 8 Peak Width /s Observed Peak Width (normalized) 50 ms 100 ms 500 ms 1000 ms 1500 ms “1.1” line with peak width at 50 ms set to “1”
- 173. 173 Conclusions Important Notice for Mobile Phases Selecting Water / Organic Solvent; Mobile Phase Preparation; Effects of Dissolved Air Important Notice for Sample Solvents Sample Solvents and Peak Shape; Theoretical Plate Number Important Notice for Column Temperature Effects of Column Temperature Control; Column Temperature and Selectivity / Peak Shape Important Notice for Detection Detection and Temperature; Detector Response