1. HPLC Applications Training
Jeff Kibby
Field Support Specialist
25-March-2010

2. HPLC Applications Training
MOBILE PHASE CONSIDERATIONS
Flammability
Toxicity
Miscibility
Viscosity
Dissolved Salts
%Organic Solvent
pH (Acid/Alkali)
Carbon Dioxide
Corrosion

3. HPLC Applications Training
Some Flammable Solvents Commonly Used
Acetonitrile
Methanol
Hexane, iso-Hexanes, Heptane, iso-Octane
Tetrahydrofuran
Toluene
Acetone
Ethyl Acetate
Isopropanol

4. HPLC Applications Training
Some Toxic Solvents Commonly Used
Acetonitrile
Methanol
Hexane
Tetrahydrofuran
Toluene
Dichloromethane (Methylene Chloride)
Chloroform
Dimethyl Sulphoxide

5. HPLC Applications Training

6. HPLC Applications Training

7. HPLC Applications Training
HPLC SOLVENTS - UV CUTOFF Burdick & Jackson solvents are arranged in order of increasing UV cutoff, the wavelength at which the solvent absorbance in a 1 cm path length cell is equal to 1 AU (absorbance unit) using water in the reference cell. UV Cutoff (nm) Acetonitrile UV 190 Pentane 190 Water 190 Hexane UV 195 Cyclopentane 198 Cyclohexane 200 Heptane 200 Isopropyl Alcohol 205 Methanol 205 Ethyl Alcohol Methoxyethanol 210 Methyl t-Butyl Ether 210 n-Propyl Alcohol 210 Trifluoroacetic Acid 210 Tetrahydrofuran UV 212 n-Butyl Alcohol 215 1,4-Dioxane 215 Ethyl Ether 215

8. HPLC Applications Training
BURDICK & JACKSON
UV Cutoff (nm)
Acetonitrile UV
190
Ethylene Dichloride
228
Water
Dichloromethane
233
Hexane UV
195
Chloroform
245
Cyclohexane
200
Ethyl Acetate
256
Heptane
Dimethyl Acetamide
268
Isopropyl Alcohol
205
DMF
Dimethyl Sulfoxide
Methanol
Toluene
284
Ethyl Alcohol
210
Trifluoroacetic Acid
1,2,4-TCB
308
Tetrahydrofuran UV
212
1,4-Dioxane
215
Acetone
330
Ethyl Ether
Methyl Isoamyl Ketone
Iso-Octane
Methyl Isobutyl Ketone
334

9. HPLC Applications Training

10. HPLC Applications Training

11. HPLC Applications Training

12. HPLC Applications Training

13. HPLC Applications Training

14. HPLC Applications Training

15. HPLC Applications Training

16. HPLC Applications Training
HPLC Columns
Silica particles (1.5~100μm)
Polymer particles (3~50µm)

17. HPLC Applications Training

18. HPLC Applications Training

19. HPLC Applications Training

20. HPLC Applications Training

21. HPLC Applications Training

22. HPLC Applications Training

23. HPLC Applications Training

24. HPLC Applications Training
SAMPLE PREPARATION
Filtration – Membrane Filters 0.2µm, 0.45µm (Cellulose Acetate, PVDF)
Centrifugation – Economical, less contamination, multiple processing

25. HPLC Applications Training
PS335 Photodiode Array Detector

26. HPLC Applications Training
PS335 Photodiode Array Detector

27. HPLC Applications Training
PS335 Photodiode Array Detector

28. HPLC Applications Training
PS335 Photodiode Array Detector

29. HPLC Applications Training
PS335 Photodiode Array Detector