chrom lc1 instrument 2014 ENG - uttera.chem.ut.ee/~ivo/Chrom/chrom_lc1_instrument_2014_ENG.pdf ·...
Transcript of chrom lc1 instrument 2014 ENG - uttera.chem.ut.ee/~ivo/Chrom/chrom_lc1_instrument_2014_ENG.pdf ·...
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High-‐performance liquid chromatography
Instrumenta8on, it’s use and maintenance
Liquid chromatography -‐ LC • At the beginning of chromatography (20th century beginning, Mihhail TsveA): – Columns 50 – 500 cm with 1 – 5 cm Ø – Eluent flow rate 0.1 ml/min – ParNcle size 150 – 200 μm
• Rise in flow rate in this system will lower the efficiency (look van Deemter).
• In order to rise efficiency and flow rate at the same Nme, it was necessary to lower the diameter of the parNcles.
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HPLC • To pump eluent to a column with small parNcles (2 – 10 μm) high pressure is needed hence HPLC.
• HPLC -‐ high-‐performance OR high-‐pressure liquid chromatography
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HPLC instruments
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Degasser
Pump
Injector/Sampler
Column
Detector
Eluents
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HPLC instrument UHPLC • To pump eluent to a column with very small parNcles (< 2 μm), very high pressure is needed – UHPLC.
• UHPLC (Ultra High Performance Liquid Chromatography) – UPLC – Ultra Performance Liquid Chromatography (Waters)
– RRLC – Rapid ResoluNon Liquid Chromatography (Agilent)
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UHPLC
• Chromatographic separaNon is the most important!
• If separaNon is sufficient, you may achieve: – higher sensiNvity – higher speed – smaller solvent consumpNon – smaller sample size
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Solvents • Organic phase:
– HPLC grade, filtrated through 0.45 μm – LC-‐MS grade, filtrated through 0.2 μm
• Aqueous phase (buffer): deionized water with resistance >18 MΩ·∙cm, it is also good to lower the content of organic compounds.
• BoAled waters may contain Na+ and K+ which may interfere with the analysis.
• Solvents cannot be stored in plas8c containers! 2014 fall 8
Eluent containers • Should be of glass (or steel) because plasNc containers leak plasNcisers and permeate gases.
• Containers must be closed with a cap (dust!), but air must be let in, eg through filter.
• Containers for aqueous solvents should be dark (amber) glass to lower the possibility for microbial growth.
• Containers must be carefully cleaned (recommended to dry at 400°C).
• Wash 10 Nmes with eluent before use. 2014 fall 9
Degasser • New systems generally have “on-‐line” degassers.
• Principle: – eluent flows through thin-‐walled PTFE-‐tube
– tube is in vacuum and gases diffuse through the wall of the tube
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Eluent
Pump
Vacuum pump
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If eluent is not degassed…
Air bubble causes an empy trace in column
Stationary phase
Most of the sample moves as supposed to through the column
Part of the sample moves faster
• Faster moving part moves faster through the column, causing a front for peaks.
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If eluent is not degassed…
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Notes on degassers • Degasser can hold as much as 20 ml of eluent. Keep in mind when changing the eluent!
• In some degassers vacuum changes largely and the efficiency of degassing is changing in Nme.
• When degasser is not used for longer period, it should be washed with deionized water (to remove salts, microbial growth) and then with methanol.
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Pump • Mostly reciprocaNng pumps, where the flow of eluent is created by the synchronized movement of piston and valves.
• For gradient eluNon, systems with mulNple pumps are used.
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Flow of eluent
ReciprocaNng pump • Pros:
• Can generate high pressure • Flow of eluent is well controlled • Possibility for gradient eluNon • Small internal volume (35 – 400 μl)
• Cons: • Flow of eluent is pulsing and it must be corrected
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UHPLC difference from HPLC
• Higher requirements for pumps and tubing connecNons.
• Outer column volume must be minimal. • Detector:
– Flow cell volume is small. – Fast detecNon speed.
• Eluents and separaNon principles are the same.
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Forming gradient eluNon • Two types of mixers:
– Low-‐pressure mixer – eluents are mixed before entering the high pressure pump
• Cheaper to buy and own – spare parts only for one pump • Dead volume of gradient is larger
– High-‐pressure mixer – for each eluent component there is a separated high pressure pump and mixing takes place aoer the pumps
• Electronics must handle the different compressibility of eluents
• Pump must take into account the volume effect of mixing (up to 20%!)
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Sample injecNon – loop • The most simple version is a valve with a loop.
– Many automaNc samplers also use the systems with loops
– Mostly the whole loop full is injected at once – sample size is fixed by the loop size, but it is possible to inject a partly filled loop (then the exact sizing is done by syringe)
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Sample injecNon – loop
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InjecNon valve
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Sample injecNon – automaNc • AutomaNc system (autosampler)
– Syringe can be staNonary or moving
Sample injecNon – automaNc • Pros and extras:
• Do not need tools for changing the sample volume • AutomaNzed • Needle washing • Sample diluNon • Mixing in needle or in vial (inject sample components from different vials eg. carrying out chemical reacNons)
– Not all autosamplers can carry out the listed operaNons
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Autosampler vials
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Autosampler – maintenance, cleaning
• Carry over
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Autosampler – maintenance, cleaning
• ContaminaNon of autosampler
Column thermostat • Allows to maintain the temperature of the column constant and use temperatures lower and higher than the room temperature.
• Systems with circulaNng air or water are available, most convenient are thermostats with PelNer' element.
• Column informaNon sheet must be checked for the highest suitable temperature. Generally over 60°C is not recommended.
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Temperature gradient in column
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Columns • Column case is usually from stainless steel with internal
diameter 2 – 4.6 mm. Outer size ¼ inches and length 10 – 30 cm.
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Column material
Proper8es Figure
Stainless steel Most widely used material. Very inert and can tolerate very high pressure (up to 1300 bar).
Titan Used when stainless stell is not inert enough. Titan-‐zirconium alloys are very strong and inert.
PEEK Tolerate pressure above 90 bar. Do not tolerate concentrated nitric and sulphuric acid, THF, DMSO.
Glass Cheap, inert. Don’t tolerate THF. First ones tolerated only 10-‐20 bar, nowadays can tolerate higher than 90 bar.
Column ends
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Guard column • Used before analyNcal column in order to lengthen the lifeNme of an analyNcal column.
• Guard column catches: • Solid parNcles (from eluent, sample) which may clog the column • Sample components which may chemically bond to the column parNcles and degrade it.
• Filling of the guard column should be the same as in analyNcal column but with larger parNcle diameter.
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Guard column
• When connecNng guard column, it should be kept in mind that the connecNons should be as short as possible – to avoid the rise in dead volume.
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Guard column? • Guard column and connecNons cause a loss in efficiency.
• Guard column and holders cost money ...
• Is it worth to use the guard column?
• There are authors who say that guard columns are a selling argument of producers.
• Everyone must calculate and test themselves
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Guard column!
• Recommended when – Samples are “dirty” – Many samples are analyzed – Analysis is at high temperature – Column pressure rises aoer short use of column – Aoer few injecNons retenNon Nmes start to shio
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Detector • MulNple detectors are used which are based on measuring different physical and chemical properNes: – UV-‐Vis absorpNon (fixed wavelength ... diode-‐array) – fluorescence – conducNvity – electrochemical detector – mass spectrometric
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Data acquisiNon • New instruments are connected to computer. Roles of computer:
• Controlling the parameters of chromatograph (flow rate, sample size, etc) and monitoring.’
• CollecNng the detector signal • Saving methods (sets of working parameters) • Data processing
• Chromatographs produce a lot of data and computers must be fast and with enough space for data.
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Tubing • For high pressure, two types of tubing are used:
– Stainless steel: • Easy to bend • Corrodes (eg. Cl-‐) • Hard to cut (or buy fixed length tubing)
– PEEK (polyether ether ketone): • Inert, swells when using THF, DMSO and methylenchloride • Hard to bend (can use special corners)
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ConnecNons • Swagelok-‐type connecNons are used in many cases.
• With Swagelok-‐connecNons cone fixes to the tubing at the first use and this cannot be changed.
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Swagelok-‐conecNon
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• NB! If tubing has been used for one producer column, it may not fit to column by other producers! • It is beAer to use rubber Nps than metal
ConnecNons • Stainless steel tubes are connected with Swagelok-‐type connecNons.
• Need tools for making connecNons
• For PEEK-‐tubing connecNons that can be used without tools -‐ finger-‐:ght nut. • Tip! Finger-‐(ght can be used on stainless steel tubing also!
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ConnecNons – recommendaNons
• Different producers may have different connecNons.
• Do not over8ghten the connec8ons! • Do not use same Swagelok’s for new connecNons. • Always use the right sized tools. • Check that connecNons are inert towards the used samples and eluents.
• Always use connecNons with the smallest dead volume.
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Convenient extras • Solvent choosing valve (pump add in, need extra degasser canal). Chromatographically does not add anything but makes working more convenient when need other solvents:
• For column washing • For other chromatographic method
• Column choosing valve (add-‐in to column thermostat) allows:
• To change column without any tools • Use different eluNng procedures (back-‐flush)
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Maintenance (cleanliness) • Cleanliness is of main importance. Solid parNcles (dust) can damage the pump seals and block the column.
• Filtrate eluents before use! • Filtrate samples before use! • Filtrate with filters with 0.4 μm diameter pores. For UHPLC, use filters with 0.22 μm pores.
• Material of filters depends on the use. For aqueous samples PVDF (polyvinylidene fluoride) is suitable. 2014 fall 42
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• Clogging of pre-‐column frit causes a rise in pressure. HPLC columns have frits (similar to filters) in both ends and protect the staNonary phase. Pore size of the frit is smaller that the parNcle size in column.
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Maintenance (cleanliness) Maintenance (cleanliness) • If eluent is not filtrated, dust and other dirt parNcles can arrive at the column and sNck to the staNonary phase.
• This dirt rises the system pressure which in turn shortens the column lifeNme.
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• To insure the clean column, it is good to use in-‐line filter before (guard) column.
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Maintenance (cleanliness) Maintenance (chemical compaNbility) • When choosing an eluent, keep in mind materials it becomes to contact with in system – Pump, seals and tubing – Column parNcles
• If eluent contains salts, wash the system with working eluent without buffer salts. Then system must be washed with organic solvent (eg. acetonitrile), to avoid corrosion.
• Some pumps have external washing possibility.
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Maintenance (column) • StaNonary phases of columns are quite durable, it is beAer to protect them from steep changes in pressure. – Change the flow rate in small increments – Change eluent composiNon in small increments
• Avoid movement of eluent in the “wrong” direcNon in the colum – Connect column the same way every Nme – When opening the column, open the tubing of the end of the column first.
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ObservaNon (external) • Listen to ...
– degasser – pump – valves (if have any)
• Look for ... – leaks – bent connecNons – visible mechanical damages
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ObservaNon (pressure) • The first indicaNon of the system is pressure. Important to observe the pressure at all Nmes! – If pressure is fluctuaNng, there is an air bubble in the system (normal fluctuaNon for binary pump at normal pressure is around 1 bar)
– If pressure is too low, it is good to look for leaks – If pressure is too high, then
• (Guard)column could be clogged • Tubing could be blocked or dented
• Pressure is a good indicator for column state.
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Instrument maintetace • Maintenance procedures are necessary when
– Some observed parameters are not normal – System does not pass tests (determined by the laboratory or instrument manufacturer)
– System notes the need for maintenance – Planned maintenance (annual, every month etc)
• If maintenance is carried out by the lab personnel, instrucNons must be followed very carefully!
• Check, if lab maintenance influences the warranty! 2014 fall 50
Column maintenance • Check column documentaNon for column use and maintenance instrucNons.
• In order to clean column from strongly adsorbed components, wash with organic component of the eluent. – Acetonitrile – Cyclohexane/acetonitrile/iso-‐propanol (25:25:50) – Dichloromethane/methanol (95:5) – In some cases low flow of DMSO or dimethylformamide
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