HPLC. GRATE Chromatography Lab Course. Dr. Johannes Ranke. September 2003

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1 HPLC GRATE Chromatography Lab Course Dr. Johannes Ranke

2 Organisation The groups Start at 9:00 am End at 18:00 pm at the latest Friday, 19th we will finish at 2:00 pm Thursday, 11th: Lecture at 08:15 am Please be prepared! Document your experiments

3 Lab journal and final report Write down what you have to do in the day Include safety measures Start collection and evaluation of data during the experiment

4 Prof. Jastorffs way Operation Observation Explanation

5 Substance information Inform yourself about the safety data of the substances you are working with (especially methanol and acetonitrile!) A collection of material safety data sheets (MSDS) will be available in the lab Use the colour coding scheme for discussing molecular interactions and predicting the elution order of your analytes

6 Some basic terms in HPLC analytes or solutes separation mobile phase (mixture of organic solvents and water) stationary phase (packed in a high pressure column) by molecular interactions

7 Characteristics of HPLC Interaction between stationary phase (small particles) and mobile phase Formation of an equilibrium distribution between the mobile phase and the stationary phase High pressure gradient within the column Small theoretical plate height, but short column Smaller separation number than GC Good for large, polar and ionic compounds (no derivatisation is needed)

8 Basic HPLC system Eluent bottle (mobile phase) Pump (max. 250 bar, flow rate: ml/min) Injection valve with sample loop (20 µl) Column (packed, porous material 5-10 µm) Detector (UV, Fluorescence, Refractometry) Integrator Eluent waste container (Please use seperate waste bottles for methanol or acetonitrile respectively!)

9 The injection valve waste sample sample loop eluent eluent to the column to the column Load position Inject position

10 The column in HPLC Inside the column:

11

12 t M = dead time Injection of a sample t R = total retention time time

13

14 Separation by means of retention Adjusted or Net retention time (t MS ) t MS =t R -t M Where t m is usually termed dead or hold up time, and is defined as the time it takes for the mobile phase to travel from the injector to the detector. The net retention time is a function of the partition coefficient of the solutes between mobile and stationary phase!

15 Separation efficiency Capacity factor k k = (t MS - t M ) / t M Resolution R R = (t 2 - t 1 ) / [0.5 * (w b1 + w b2 )] Number of theoretical plates N = 8 ln 2 (t MS /w 0.5 ) 2 Height equivalent of a theoretical plate (HETP) H = L / N with L= length of the column

16 The lab course - experiments Preparation and degassing of the eluents Operating the device Determination of dead time and dead volume Determination of column data Optimising separation of camp metabolites

17 Preparation of the eluents Deionised water Document how you prepared the eluents (which solvent, solvent/water ratio?) Learn how to degas

18 Operating the device Pressure vs. flow rate Get a stable base line

19 Determination of dead time 0.1 % acetone Dependence on the flow rate (t M and V M ) Dependence on the injection volume Dependence on the acetone concentration?

20 Determination of column data Toluene, Phenol, Anisol Linear regression for: log k = a Ø + b for each compound. Ø is the volume fraction of methanol in the mobile phase

21 camp: words inside the cell camp: Cyclic adenosine monophosphate Synthesized inside cells from the nucleotide AMP (also found in nucleic acids like DNA!) The enzyme adenylate cyclase forms the camp at the cell membrane camp acts as one of the central cell signaling molecules words regulating important cell functions Formation and degradation are strictly regulated metabolites are of biochemical interest

22 Separation of camp metabolites Colour code the chemical entities according to Prof. Jastorffs scheme Please predict the elution order of all camp derivatives Think about the influence of the TEAF buffer in your eluents Optimisation of the eluent mixture

23 Metabolism of camp in the liver Reproducible injection is essential Present kinetics of camp degradation graphically Calculate kinetic constant in s -1 ln (c (t) /c 0 ) = - k t Calculate half-life t 1/2 = ln 2 / k

24 Preparation Come prepared! Follow the instructions in the Preparation document Collect MSDS information If you have any questions, please ask.

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