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

Aims Achieving maximum performance Achieving reproducibility High speed

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

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

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 http://www.idex-hs.com/support/upchurch/i/fittings/figure3b.gif http://webstore.idex-hs.com/TechInfo/spd1Eng.asp

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

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

Unions Unions are source of dead volume Use Zero dead volume (ZDV) unions Or a type with same or smaller diamter than the tubing http://www.vici-jour.se/images/zdv.gif

A useful webtool http://webstore.idex-hs.com/TechInfo/conversions.asp

Low pressure fittings (usually 1/8 inch tubing) Usually flanged fittings, ¼ inch 28 tpi Or the flangeless version with Inverted ferrules http://www6.gelifesciences.com/Images/mediumres/18465201-01.jpg Or the ingenious M6 from Pharmacia

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

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

Not bad

problems

More problems

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)

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

A typcial calibraion curve http://www.sagewisdom.org/phytochemicalfig2.gif

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

Inject samples

Get results

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

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 http://www.labcompliance.com/tutorial/methods/default.aspx#01_Intro

Method validation and system suitability http://www.cvg.ca/images/SystemStabilityTests.pdf

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

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)

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)

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

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

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

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

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

SPE rack Different forms, Other examples: - pipette tips centrifugecartridges, Disposable columns http://en.wikipedia.org/wiki/File:SPE_Manifold.jpg

http://www.waters.com/waters/nav.htm?cid=10083845

Mixed SPE resins http://www.waters.com/waters/nav.htm?cid=513209

http://www.waters.com/waters/nav.htm?cid=10083883