1. Setting up, testing and running LC-systems
Ulrich Bergmann
2009

2. Aims Achieving maximum performance Achieving reproducibility
High speed

3. Performance
Maximum performance achievable is determined by the column with its theoretical plates
Better it can’t be, but it’s easily possible to loose and get results far below the theoretical plates theoretical possible.
Reasons lie in
System set up
Type of sample and sample application

4. System set up Avoid dead volumes Components of suitable size
Flow cell in detector
Pump head
Mixer
Sample loops
Size of tubing
Correct plumbing

5. HPLC connections
Pressure proof up to 100 bar, depending on type and material
Standard are 1/16 inch
Tubing (nano also 1/32),
The thread is UNF gauge 10
(3/16 inch (4.76 mm) with 32
Threads/inch
The tubing should fit tightly
Into the fitting and is sealed with a conical ferrule (press fitting)
The tube has to reach the bottom, otherwise dead volume (mixing chamber)-
If ferrule sits too high, leakage

6. In real life
Take the fitting with loose ferrule, push tubing to the bottom of the fitting, hold it and then tighten the screw
Plastic (PEEK) fittings are open again when released, meaning they can be reused in another connection
Steel fittings crimp and are then fixed, they can only be used in the same type of fitting, or better, only in the one and only connection they have been made for.
Different manufacturers (can) have different ferrule shape, test and take care

8. Tube cutting
Tube ends should be square and smooth, with uncompromised opening
Comparably easy for plastic tubes
Not that easy for steel tubing
Use ready tubing, or special equipment for cutting

10. Unions Unions are source of dead volume
Use Zero dead volume (ZDV) unions
Or a type with same or smaller diamter than the tubing

11. A useful webtool

12. Low pressure fittings (usually 1/8 inch tubing)
Usually flanged fittings, ¼ inch 28 tpi
Or the flangeless version with
Inverted ferrules
Or the ingenious M6 from Pharmacia

13. Plumbing is ready, the first test:
Replace column with ZDV-union, flow 1 ml/min
Inject 2-5ml easy detectable sample
Measure peak width
Should be around 100ml

14. Second test Reproducibility
Probably the most important featur of a Chromatographic method.
It is important to know, how precisely a system can reproduce conditions
Rund gradients, preferably step gradients, with spiked solvents
B and D with 0.1% acetone, 280 nm
no column

15. Not bad

16. problems

17. More problems

18. If system works Controls Check reproducibility
Negative controls (blanks)
No column
No sample
0 injection
Injection with sample solvent only
Identify and registrate ghost peaks
Positive controls (standards)
Test samples with known properties
Check reproducibility
Retention times
Signal strength (integrals)

19. Quantitation and calibration
Need to know the response factor for each component to be quantified
Apply different amounts and record detector singals
Create calibration curve
Modern chromatography software has automatic routines

20. A typcial calibraion curve

21. Automatic quantitation
For automatic quantitation you need to
Define retention time limits
Integration criteria (define how peak-start and
-end is determined)
Provide concentrations
Rund standards

23. Inject samples

24. Get results

25. Routine operation contain sample sets with
Standards
Blanks
Controls
And unknowns
To proof reproducibility, accuracy, and precision of the method

26. Method validation and system suitability
In many cases chromatographic methods have to be approved by authorities and the method has to be suitable for the task in question

27. Method validation and system suitability

28. If something is wrong! Trouble shooting
Analyze and isolate the problem
But don’t fall in love with your nice theories
Observe carefully
Flow at end of system
Pressure
Change only one parameter at a time

29. Example 1: high backpressure
Loosen fluid connections strategic points
Find the bad guy
Clean (ultrasonic, reverse flow)
Replace or repair faulty parts (rare except for columns and filter frits)

30. 2. Example: unregular flow
Observe pressure
If fluctuating, check for air in system, degass and purge if necessary
Check valves
The most usual reason for pulsing, flow is unsteady and apparently comes only from one pump head.
Put check valves in methanol and ultrasonic for 30 min
Replace if necessary (not very likely)

31. Check valve diagnosis
Disconnect one pump head and plug its connection.
Plug the system behind pressure sensor
Put a low flow rate (and a reasonable pressure limit) and wait for pressure to rise. If so, inlet check valve OK, repeat with the other pump head.
Reconnect both pump heads and start again low flow. When pressure breaks down, the pump head pulling back has a faulty outlet valve

32. Leakage test Put decent pressure limit!!!!
Block system after the component to test.
Start low flow
If pressur limit not reached, bingo.
If it is blown, observe pressure decrease

33. Unusual poor resolution Unusual poor resolution
Old column
Fluid connections
Something wrong with the sample

34. What can be wrong with sample? Sample preparation
Sample solvent
Should be of lower elution strength than the mobile phase
at least when larger volumes are applied and/or in gradient methods
Sample volume
Critical in isocratic appliations and/or if sample is dissolved in a solvent of higher elution strength than the mobile phase
Sample matrix
This is the fancy word to describe all the other stuff that might be in the sample and can effect the chromatographic behaviour

35. SPE
To get some control over the sample matrix, very often a prepurification is done before the chromatographic step.
Compound of interest can be in flow through or retentate.
The latter is more common since it allows volume reduction
This usually employs adsorption chromatography using similar methods than ”real” chromatography techniques.
Group selective separation with high capacity
However, many SPE is a mixed separation

36. SPE rack Different forms, Other examples: - pipette tips
centrifugecartridges,
Disposable columns

38. Mixed SPE resins