Welcome to Class!

1. Welcome to Class! Kevin Olsen 359 Richardson Hall 973-655-4076 OlsenK@Mail.Montclair.Edu

2. Class Expectations and Grading • Grades will be awarded based on laboratory reports and homework assignments. There may be quizzes given in class depending on time limitations. • Some homework assignments will require an oral presentation to the class. • Unless specifically stated otherwise, the standards of documentation required on laboratory reports will be those found in a GMP / GLP regulated environment.

3. Laboratory Reports • All paperwork is a permanent record. Nothing will be discarded. • All chromatograms will include: • Student name • Date • Sample ID • Chromatographic conditions • System ID number and calibration dates

4. Laboratory Reports (continued) • Laboratory reports will be checked by a second person before being turned in. The person checking the report will sign it. • Any cross outs will consist of a single line, initials and date. In some instances such as a faulty injection, an explanation for the cross out will be included. • No records will be kept in pencil.

5. Laboratory Reports (continued) • The identity and calibration data of each and every instrument used will be included on the laboratory report, this includes balances and pH meters. If an instrument is not calibrated, that fact will be noted. • The manufacturer and lot number of each reagent used will be recorded. (Mobile phases are exempt from this rule.)

6. The Cardinal Rules of GMP / GLP • If it was not documented, it did not happen. • To err is human, to destroy the evidence is a felony. • Maintain every record as if it were going to be reviewed by the FDA. • Your signature is precious, never sign, approve, or authorize anything you are not absolutely sure is correct.

7. Lesson 1 • Review of HPLC components • Operating your HPLC

8. A Generic HPLC System

9. Generic HPLC Pump

10. Controlling your HPLC Pump • Flow rate • Minimum and maximum pressure • Purge valves • Mobile phase mixtures

11. Injector

12. Variable Wavelength & Photodiode Array Detectors

13. Column Heaters Some separations of complex mixtures must be temperature optimised to achieve separations of overlapping peaks. Increased column temperature will also shorten retention times for a given column dimension. Selectivity of the column is also a function of temperature and elution orders of peaks may change and even reverse - some chiral and amino acid separations are very sensitive to column temperature effects. Eliminates retention time variation due to room temperature fluctuations.

14. Column Heaters

15. Bits & Bobs

16. Bits and Bobs

17. If Your Fittings Leak • Check to make sure your tubing is seated properly • The fitting may not be tightened enough • You may be using incompatible fittings • Check the condition of the nut and ferrule • Sometimes a leaking connection has nothing at all to do with the nut and ferrule, but with the receiving port • NOTE: Using fittings made of material that is incompatible with your mobile phase is a sure way of creating leaks

18. Bits & Bobs

19. Lesson 2 Running a simple separation

20. “Normal Phase” + - + - + - + - + - + - + - + - + - + - + - + Mobile Phase -NON POLAR + - + - + - + - + - + - + - + - + - + - + - + Stationary Phase - POLAR

21. “Reverse Phase” Mobile Phase - POLAR + - - - + + Stationary Phase - NON POLAR

22. Reverse Phase Silica Stationery Phase support The longer the alkyl chains, the longer the retention time in a reversed phase column.

23. Why Degas the Mobile Phase? Actual nitrogen concentration Theoretical saturation 100% water-----------------------------------------------100% Methanol

24. What is a Solvent Front?

25. How to Degas the Mobile Phase • Sonicate. • Apply partial vacuum while agitating. • Helium sparge. • In-line degasser.

26. Gradient Ethyl acetate Methanol - ammonium acetate Acetonitrile - water 20 minutes

27. Things to try…. • Mobile phase composition. What happens to retention times and peak width? • Pump speed. What happens to peak width? • Column temperature. What happens to retention times? • For Homework: Prepare a written report explaining your findings and include neatly tabulated data. Calculate column efficiency using the same analyte on each first run. You do not have to submit your chromatograms.

28. Homework Report Format

29. Peak Width at Half-height • Peak width, or sharpness, is an indication of column efficiency. • An ideal peak is a Gaussian distribution. • The ratio of standard deviation to retention time is independent of flow rate. tr

30. Calculating Column Efficiency • Peak width at half height is used to calculate column efficiency. • N = 5.54 ( T / W )2 Where N = theoretical plates W = Width at half height T = Retention time 5.54 is a constant based on the normal distribution

31. Lesson 3 Changing Columns Sample Loops Care and Maintenance of Injectors

32. Installing a Sample Loop

33. Never use a pointed or bevel tip needle. Rinse after the use of buffer solutions. Avoid abrasive particles by filtering samples before injection. Use burr-free tubing to avoid metals shavings from getting into the injector. Routine Care of Injectors

34. Routine Care (continued)

35. Leaks between stator and stator ring? Leaks in needle port ONLY when loading? Fluid escapes by siphoning out? Continual leak out needle port or vent lines? Loosen set screws or tighten stator screws. Tighten needle seal by pushing in the guide. Adjust the height of the outlet tubes. Surface of the rotor is scratched, replace. Leak Checking the Injector

36. Load Volume • For a partial fill, use no more than 50% of the loop volume. The sample liquid tends to expand and fills the loop completely. • To completely fill the sample loop, use at least 200% of the loop volume.

37. Flushing the Needle Port Tube • Use the manufacturer’s recommended adapter and connect it to a luer lock type syringe. • Flush in the INJECT position only so that the fluid flows out the vent line. • The sample loop will be flushed with mobile phase.

38. How Do I Know My Injector is Clogged?

39. Flushing the Stator and Stator Face • With the pump turned off, disconnect the one sample loop fitting and the column connection. • Using a syringe, gently pump a cleaning fluid (high purity methanol or water) through the ports.

40. Lesson 4 Care of HPLC Pumps

41. Pump Care • Flush with water after running a buffer, (note there are special procedures when using reverse phase columns.) • Replace seals in a timely manner. • Maintain check valves. • Do not allow solids in the mobile phase.

42. Removing Buffers from a Reverse Phase Column • DO NOT FLUSH WITH 100% WATER AS YOUR FIRST STEP - • Substitute water for the buffer but leave the remaining proportions the same. Run through about 5 column volumes. • Wash through 10 column volumes of a strong organic solvent, example - Methanol. • If you plan to store the column, read the directions. • If the phase collapses, a 50-50 water, organic solvent wash for 30 minutes can restore it.

43. Lesson 5 HPLC Calibration

44. Why Calibrate? From an FDA Warning Letter of June 29, 2002 issued to a Medical Gas supplier: • 3. Failure to routinely calibrate mechanical and electronic equipment or keep records of calibration according to a written program designed to assure proper performance as required by 21 C.F.R. 211.68. For example, your electronic thermometer was last calibrated in June 1999, the pressure gauge for the manifold filler was last calibrated in February 1997, and the vacuum gauge has never been calibrated.

45. Why Calibrate? From an FDA Warning Letter of June 29, 2004 issued to a company that makes metals used in dental implants: …not in conformity with the Current Good Manufacturing Practice (CGMP) requirements of the Quality System (QS) regulation found at Title 21, Code of Federal Regulations (CFR), Part 820. Significant violations include, but are not limited to, the following: 1. Failure to establish and maintain procedures to ensure that equipment is routinely calibrated, inspected, checked, and maintained, as required by 21 CFR 820.72(a). For example, your written procedures require that equipment be calibrated two times a year. The following equipment has not been calibrated or checked two times a year: the…machine used to test the yield strength, ultimate tensile strength, and elongation on the has not been calibrated. The next calibration date was supposed to have been January 28, 2004. 2. Failure to document the equipment identification, calibration dates, the individual performing each calibration, and the next calibration date, as required by 21 CFR 820.72(b)(2). For example, the calibration data sheet dated August 26, 2003, for the spectrophotometer used to analyze other metals was not signed and dated by the individual who performed the calibration and had no specified test value for the “Std. Dev. Sample Units”.

46. Calibration • wavelength accuracy, • wavelength precision (repeatability) • absorbance linearity • absolute absorbance accuracy.

47. Automated Detector Calibration Filter with a known Absorbance(s) Flow cell To waste From column

48. Automated Detector Calibration Filter with a known Absorbance(s) Flow cell To waste From column

49. Holmium Oxide

50. Agilent 1100 Wavelength Calibration 486 nm 656.1 nm 1 nm slit Deuterium lamp