HPLC troubleshooting for changing baseline, tailing peaks, and pump and detector problems.
3 Troubleshooting Case Study A Few Clicks to Find the Probable Cause Using the problem of negative peaks described above, the HPLC Troubleshooting …
Nov 09, 2013 · Reverse phase chromatography is the most commonly used LC or HPLC separation mode.It is used to separate nonpolar molecules in solution.In reverse phase the stationary phase is nonpolar and the mobile phase is polar.The name “reversed
Nov 01, 2013 · normal phase liquid chromatography, polar, stationary phase, nonpolar, table i.1, properties, mobile s phase, retained solute, order of elution, what is …
HPLC Troubleshooting Guide. 2 www.ace-hplc.com Quick Reference Tables If you have a problem you need to solve now, use these Troubleshooting Tables. ... Check and adjust pump flow rate (see 2.4) Column overloaded 1. Reduce amount of sample injected 2
compensation software which is found in most modern HPLC systems. Many of these systems will allow you to manually enter the actual liquid compressibility values for each liquid (pump channel) used. This can result in better baseline stability and le
3 Figure B. Supelguard Columns Prolong the Lifespan of Your Analytical Columns column: SUPELCOSIL LC-PCN, 25 cm x 4.6 mm I.D., 5 μm particles (with
A Troubleshooting Guide – Version 1.1. 2 1. Introduction 4 1.1 Purpose 4 1.2 Content 4 2. ... system pressure under normal operating conditions each day or each time your HPLC is used. ... pump manual. 5. Loosen detector waste outlet ﬁtting.
HPLC Column Selection Guide Column Parameters Internal Diameter An important parameter of a HPLC column is the internal diameter (ID) as this directly influences the
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HPLC Solvent Properties Introduction for non-chemists Organic solvents fall into classes, such as alkanes, alcohols, acids, aldehydes, etc where each member of the class has the same functional group, e.g. OH (-alcohol) Each member has a different alkyl chain length (=the number of carbon atoms in the molecule joined is a chain.). This then has a standard naming system (nomenclature). No Functional Group Methane, CH4 Ethane, CH3CH3 Propane, CH3CH2CH3 Butane Pentane Hexane Heptane Octane Nonane Decane
Functional Group Methyl, CH3Ethyl, CH3CH2Propyl, CH3CH2CH2
(NB 18 carbons – octadecane = ODS) When the carbons are all in a straight line, it is prefixed by ‘n’ e.g. n-pentane, n-hexane etc. Some solvents have more than one name because different naming systems have been adopted. For example acetic acid, CH3COOH, is also known as Ethanoic Acid (2 carbons). Isopropyl alcohol is also Propan-2-ol, iso-octane is also 2,2,4 trimethyl pentane and methyl ethyl ketone is also known as butan-2-one. Function groups determine the rest of the name:
-OH adds –ol (ie alcohol, e.g. methanol, ethanol etc) -CN nitrile e.g. Acetonitrile -NH2 amino -C6H5 Phenyl
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Solvent Parameters relating to HPLC UV Transparency and UV cut-off Most solvents are more transparent to UV down to a certain wavelength and below that they totally absorbs UV so the spectrum looks as follows:
To be useful with UV detection, the solvent has to have a lower UV cut off then the absorption of any of the sample components. In general, reverse phase eluents have much lower UV cut-off’s than normal phase eluents.
Solvent Miscibility Some solvents such as alkanes (e.g. hexane, pentane etc) are very non-polar and will not mix with others (such as water) which are very polar. Since solvents are mixed in HPLC to fine tune the polarity, thereby controlling their eluent strength, it is essential that solvents chosen are totally miscible. Also when changing from one solvent to another, the new solvent must be miscible with the old one. If this is not the case, a stepwise change must be made. See table for miscibility.
Viscosity HPLC operates in dynamic equilibrium. Almost 90% of the surface area a 5u packing material is inside the pores. So the lower the viscosity of a solvent, the lower the back pressure and the better the mass transfer in and out of the pores. This in turn gives better separation efficiency, ie sharper peaks. See the table for viscosity of most HPLC solvents.
Purity In general lab use we are familiar with AR, LR and technical grades. For HPLC we require a higher level of purity. However, just as different manufacturers spec for Analytical Reagent (=AR, Analar) and Laboratory Reagent (=SLR, GPR etc) varies, so does ‘HPLC Grade’. For the purpose of this, our HPLC grade starts out as AR grade raw material, following which it is distilled at least once and filtered. Some manufacturers test only the optical purity (ie UV transparency) and hence it does not even meet AR spec. The importance of solvent purity is that when analysing 20ul of sample, looking for possible sub picogramme levels of sample, impurities in the 20-30ml of solvent used during a run can be quite significant. Some solvents (such as heptane) are very expensive in pure form and 95 –97% may have to be used. However, impurities are very similar alkanes and normally have little effect on the separations.
Stability Most solvents are stable for long periods. This is indicated by their shelf-life unopened. Some solvents, especially THF, have very limited shelf life, so it is important only to order as many as can be used within this period and to ascertain the confidence level that can be placed in the integrity of a bottle of solvent retrieved from a solvent cupboard. Look for Date of Manufacture, and shelf life.
Eluent Strength For Reverse Phase HPLC, water is the weakest eluent. It’s eluent strength is then modified by adding a less polar but miscible solvent such as methanol. The less polar, the greater the eluent strength. For Normal Phase HPLC, hexane (or heptane) is the weakest eluent and a more polar solvent is added to modify eluent strength. These include Chloroform, Dichloromethane, Ethyl Acetate, Acetone, Ether etc If a change in eluent composition is made for a selectivity reasons e.g. from Methanol to Acetonitrile in RP-HPLC, the ratios must be changed to maintain the same eluent strength.
COSHH Some solvents are more hazardous than others. Look for toxicity, flammability , carcenogenicity etc. Some have a very unpleasant odour. Some have a low flash point. It is important to be aware of the hazards.
Compressibility Compared to gases, solvents are not compressible. But compared to each other, some are more compressible than others. Some pumps have the facility to correct for this, thereby achieving a more accurate flow rate (See table.)
Refractive Index Light is refracted (a deviation from the angle of incidence, ‘bent’ to the geography teachers) when it passes from one medium to another, e.g. from air into a liquid, or into glass. The refractive index of a material is an index of the extent of this deviation. Taking air as 1.0, water is 1.3 and glass is 1.5. A refractive index detector operates by measuring a change in refractive index when a sample component passes through the flow cell. It is therefore important that the RI of the sample and of the eluent are as different as possible. Otherwise no peaks! See table.
Cost Some solvents are very much more expensive than others. The range is from about £7 per 2.5 litre Winchester to over £100 per bottle. Disposal may be expensive
A few specific solvents Chloroform – Should be stabilised, 0.5% Ethanol. Unstabilised is dangerous. Especially with Aluminium. THF – Unstabilised has 6 months. Stabilised 2 years. Do not distil (contains free radicals). Stabiliser is BHT. (Not miscible with water, strong UV absorption). Water - De-ionised to 18MW. Distilled with KMnO4 to oxidise and remove organics. Filtered. Very viscous. Hexanes – n-hexane, iso-hexane, hexane fraction. 95%, 97%, 99% Pet Spirit (also called Pet Ether.) Boiling factions 30-40, 40-60, 60-80, 80-100, 100-120oC Ethanol – Not a good eluent. £49 duty per Winchester (2.5litres) IPA – Excellent air solubility. Mixes with almost everything. Acids – Use acetic, formic or phosphoric.