LC Method Development and Transfer

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Erica Scott
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1 LC Method Development and Transfer I would like to reduce solvent usage and waste generation for our lab s analyses and methods. Which type of column should I choose? The 3.0 mm ZORBAX Solvent Saver ( micron) and Solvent Saver Plus (3. micron) columns are excellent choices not only for decreasing solvent and waste disposal costs, but also for providing significant advantages for LC-MS and other applications where 2-3 fold improvements in detection and quantitation limits are sought. Moreover, almost all of these columns (except those < mm long) can be used effectively with flow cells (13 ul for 1 DAD, 14 ul for 1 VWD, 8 ul for FLD) and tubing (0.17 mm, i.d.) that are sold standard with Agilent 1 systems. Click here for additional information. I'm developing a method by reversed-phase HPLC. Do you have a strategy or any recommendations? There are a number of different approaches that can be taken for RPLC method development. Conventional wisdom from many HPLC experts currently suggests that method development begin at low ph (ph 2-3.), using either a C8 or C18 column. They also recommend that a true conjugate acid-conjugate base type buffer be used, for example, NaH2PO4/H3PO4, at a concentration of 20-0 mm. Other additives that can be used to control ph include, but are not limited to: % v/v trifluoroacetic acid (TFA), % formic acid, 0.1-1% acetic acid, and ph-adjusted water using phosphoric acid (maximum ph 2.9 to ensure good ph control). Please refer to our small molecule method development guidance for more information. It is important to remember that method development and some type of method validation should always be done concurrently, as the importance, scope, and/or use of the method increases. Method validation can begin early in method development with simple tests of selectivity and injection and analysis repeatability. What are recommended Agilent HPLC columns suitable for most LC methods listed with the USP? he US Pharmacopeia (USP) is a standard source for many pharmaceutical methods. The USP specifies columns by packing materials rather than by manufacturer. Listed below are the recommended Agilent Technologies HPLC columns suitable for most LC methods listed with the USP. Click on the column phase for additional information and ordering. L1 L2 USP Packing Materials Columns Particle Size (µm) Octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 3 to 10 µm in Octadecyl silane chemically bonded to silica gel of a controlled surface porosity that has been bonded to a solid spherical core, 30 to 0 µm in ZORBAX Eclipse Plus C18 ZORBAX Eclipse XDB-C18 ZORBAX SB-C18 ZORBAX Rx-C18 ZORBAX Extend-C18 ZORBAX ODS ZORBAX ODS Classic LiChrosorb RP-18 LiChrospher RP-18 Purospher RP-18/-e Superspher RP-18/-e Nucleosil - C18 3., 3., 3., 3., 3,, 10 4 L3 Porous silica particles, to 10 µm in ZORBAX SIL Pore Size (Å) 9 120

2 L4 L L6 L7 Silica gel of controlled surface porosity bonded to a solid spherical core, 30 to 0 µm in Alumina of controlled surface porosity bonded to a solid spherical core, 30 to 0 µm in Strong cation-exchange packing: sulfonated fluorocarbon polymer coated on a solid spherical core, 30 to 0 µm in Octyl silane chemcially bonded to totally porous microsilica particles, to 10 µm in. L8 An essentailly monomolecular layer of aminopropyl-silane chemcially boneded to totally porous silica gel support, 10 µm in. L9 10 µm irregular, totally porous silica gel having a chemically bonded, strongly acidic cation-exchange coating. L10 Nitrile groups chemically bonded to porous silica particles, 3 to 10 µm in. L11 Phenyl groups chemically bonded to porous silica particles, to 10 µm in. L12 Strong anion-exchange packing made by chemically bonding a quaternary amine to a solid silica spherical core, 30 to µm in L13 Trimethylsilane chemically bonded to porous silica particles, 3 to 10 µm in. L14 Silica gel 10 µm in having a chemically bonded, strongly basic quaternary ammonium anion-exchange coating. L1 Hexyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in L16 Dimethyl silane chemically bonded to ZORBAX Rx-SIL LiChrospher 60 Si Custom Order ZORBAX C8 ZORBAX Eclipse Plus C8 ZORBAX Eclipse XDB-C8 ZORBAX SB-C8 ZORBAX Rx-C8 LiChrosorb RP-8 LiChrospher RP-8 LiChrospher RP select B ZORBAX NH 2 LiChrospher NH 2 ZORBAX SCX SynChropak SCX ZORBAX CN ZORBAX SB-CN ZORBAX Eclipse XDB-CN LiChrospher CN ZORBAX Phenyl ZORBAX SB-Phenyl ZORBAX Eclipse XDB Phenyl Accubond Bulk SAX, 2 g bottle 3., 3., 3., 3., 3., spherical 6. 3., 3., 3., 3., ZORBAX TMS ZORBAX SAX SynChropak SAX 6.

3 totally porous silica particles, 3 to 10 µm in. L17 Strong cation exchange resin consisting of sulfonated cross-linked styrenedivinylbenzene copolymer in the hydrogen form, 7 to 11 µm in. L18 Amino and cyano groups chemically bonded to porous silica partices to 10 µm in. L19 Strong cation exchange resin consisting of sulfonated cross-linked styrenedivinylbenzene copolymer in the calcium form, 9 µm in. L20 Dihydroxypropane groups chemically bonded to porous silica particles, 3 to 10 µm in L21 A rigid, spherical styrene-divinylbenzene copolymer, to 10 µm in. L22 A cation group resin made of porous polystyrene gel with sulfonic acid groups, about 10 µm in size. L23 An ion exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups, about 10 µm in size. L24 A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface, 32 to 63 µm in. L2 Packing having the capacity to separate compounds with a MW range from to 000 da (as determined by polyethylene oxide), applied to neutral, anionic and cationic water-soluable polymers. L26 Butyl silane chemically bonded to totally porous silica particles, to 10 µm in. L27 Porous silica particles, 30 to 0 µm L28 A multifunctional support, which consists of a high purity, Å, spherical silica substrate that has been bonded with anionic (amine) functionality in addition to conventional reversed-phase C8 functionality. L29 Gamma alumina, reversed phase, low carbon percentage by weight, aluminabased polybutadiene spherical particles, µm with a pore of LiChrospher Diol PL aquagel-oh AccuBond Bulk SIL, 2 g bottle

4 80Å. L30 Ethyl silane chemically bonded to a totally porous silica particle, 3 to 10 µm in L31 A strong anion-exchange resinquaternary amine bonded on latex particles attached to a core of 8. µm macroporous particles having a pore size of 2000Å and consisting of ethylvinylbenzene cross-linked with % divinylbenzene. L32 A chiral ligand-exchange packing- L- proline copper complex covanlently bonded to irregularly shaped silica particles, to 10 µm in. L33 Packing having the capacity to separate proteins by molecular size over a range of 4,000 to 400,000 daltons. It is spherical, silica-based, and processed to provide ph stability. L34 Strong cation exchange resin consisting of sulfonated cross-linked styrenedivinylbenzene copolymer in the lead form, 9µm in. L3 A zirconium-stabilized spherical silica packing with a hydrophilic (diol-type) molecular monolayer bonded phase L36 L-Phenylglycine-3, -dinitrobenzoyl on µm amino propyl silica. L37 Polymethacrylate gel packing having the capacity to separate proteins by molecular size over a range of 2,000 40,000Da MW L38 Methacrylate-based size exclusion packing for water-solubles L39 Hydrophilic polyhydroxymeth-acrylate gel of totally porous spherical resin L40 Cellulose tris-3,-dimethylphenylcarbamate coated porous silica particles, to 20 µm in L41 Immobilized alpha-acid glyco-protein on spherical silica particles, µm in L42 Octlysiilane and octadecylsilane groups chemically bonded to porous silica particles L43 Pentafluoropehnyl groups chemically bonded to silica particles to 10 µm in ZORBAX GF ZORBAX GF-20 ZORBAX GF

5 L44 A multifunctional support, which consists of a high purity, 60Å spherical silica substrate, that has been bonded with a cationic exchanger, sulfonic acid functionality in addition to a conventional reversed phase C8 functionality. L4 Beta cyclodextrin bonded to porous silica particles, to 10 µm in. L46 Polystyrene/divinylbenzene substrate agglomerated with quarternary amine functionalized latex beads, 10µm in. L47 High capacity anion exchange microporous substrate, fully functionalized with a trimethyl-amine group, 8 µm in. L48 Sulfonated, cross-linked polystyrene with an outer layer of submicron, porous, anion-exchange microbeads, 1 µm in. L49 Amylose tris-3, -dimethylphenylcarabamate-coated, porous, spherical, silica particles, to 10 µm in. L0 A strong cation exchange resin made of porous silica with sulfopropyl groups, to 10 µm in. L1 A reversed-phase packing made by coating a thin layer of polybutadiene on to spherical porous zirconia particles, 3 to 10 µm in. L2 Multifunction resin with reversed-phase retention and strong anion-exchange functionalities. The resin consists of ethylvinyl-benzene, % cross-linked with divinylbenzene copolymer, 3 to 1 µm in, and a surface of not less than 30m 2 /g, substrate is coated with quarternary ammonium functionalized latex particles consisting of styrene cross-linked with divinylbenzene. L3 An anion-exchange resin consisting of rigid, spherical styrene-divinylbenzene copolymer with trimethylammonium groups at a loading of about 2meq per g, 3 to 29 µm in. L4 Strong cation-exchange resin consisting of sulfonated cross-linked styrenedivinylbenzene copolymer in the sodium form, about 7 to 11 µm in. L Weak cation-exchange resin consisting of ethylvinylbenzene, % cross-linked with ChiraDex Chiral ZORBAX SCX SynChropak SCX

6 divinyl-benzene copolyme, 3 to 1 µm in. Substrate is surface grafted with carboxylic acide and/or phosphoric acid functionalized monomers. Capacity not less than 00 µeq/column. L6 Propyl silane chemically bonded to totally porous silica particles, 3 to 10 µm in L7 A chiral-recognition protein, ovomucoid, chemically bonded to silica particles, about µm in, with a pore size of 120Å SB-C3 3., 80 Ultron ES-OVM 120 What is the procedure for determining the Dwell Volume of my HPLC System? Procedure For Determining the Dwell Volume of Your System: 1. Replace column with short piece of HPLC stainless steel tubing. 2. Prepare mobile phase components ==>A. Water- UV-transparent ==>B. Water with 0.2% acetone - UV-absorbing 3. Monitor at 26 nm 4. Adjust attenuation so that both % A and % B are on scale. Run gradient profile 0 - % B/10 min at 1.0 ml/min 6. Record How to select buffers for my HPLC separations? Buffer Selection Buffer pk a ph Range UV Cutoff (A > 0.) Trifluoroacetic acid << 2 (0.) nm (0.1%) KH 2 PO 4 /phosphoric acid <200 nm (0.1%) Tri-K-Citrate/hydrochloric acid nm (10mM) Potassium formate/formic acid nm (10 mm) Tri-K-Citrate/hydrochloric acid nm (10mM) Potassium acetate/acetic acid nm (10mM) Tri-K-Citrate/hydrochloric acid nm (10mM) Ammonium formate (0 mm) Bis-tris propane HCI/Bis-tris propane nm (10mM) Ammonium acetate (0 mm)

7 KH 2 PO 4 / K 2 PO 4 / < 200 nm (0.1%) Tris HCI/Tris nm (10 mm) Bis-tris propane HCI/Bis-tris propane nm (10mM) Ammonium hydroxide./ammonia nm (10mM) Borate (H 3 BO 3 /Na 2 B 4 O 7 10 H 2 O Glycine HCI/glycine methylpiperidine HCI/1- methylpiperidine nm (10 mm) Diethylamine HCI/diethylamine Triethylamine HCI/triethylamine < 200 nm (10 mm) Pyrollidine HCI/pyrollidine How to prepare buffers for my HPLC separations? Buffer Preparation: Dissolve salt in organic-free water in 1- or 2-L beaker. Use appropriate volume to leave room for ph adjustment solution. Equilibrate solution to room temperature for maximum accuracy. Calibrate ph meter. Use 2-level calibration and bracket desired ph. Use appropriate audit solution to monitor statistical control (for example, potassium hydrogen tartrate, saturated solution, ph = 3.6). Adjust salt solution to desired ph. Minimize amount of time electrode spends in buffer solution (contamination). Avoid overshoot and readjustment (ionic strength differences can arise). Transfer ph-adjusted buffer solution quantitatively to volumetric flask, dilute to volume, and mix. Filter through 0.4 um filter. Discard first 0 ml filtrate. Rinse solvent reservoir with small volume of filtrate and discard. Fill reservoir with remaining filtrate or prepare premix with organic modifier. o Agilent Solvent Filtration Kit, 20-mL reservoir, 0-mL flask, Agilent PN o Nylon filter membranes, 47 mm, 0.4 um pore size, Agilent PN

Why Filtration of Contaminants in mobile phase and sample is important for HPLC? Filtration of Contaminants Frits, also referred to as filters, remove contaminants.

8 Why Filtration of Contaminants in mobile phase and sample is important for HPLC? Filtration of Contaminants Frits, also referred to as filters, remove contaminants. Within HPLC in order to achieve ultra sensitive results, it is vital to start with a clean mobile phase. There are a number of methods required to obtain a clean mobile phase. As you know, only very pure solvents should be used for the HPLC. Solvent impurities can result in baseline shifts and detector noise. Impurities will contaminate collected fractions and, if present in the weak eluent of a gradient separation, can cause ghost peaks in the chromatogram. External Filtration Considering the time and effort required producing pure solvents, the cost of commercial HPLC grade solvents is usually justified. However, all eluents, even those made from HPLC grade solvents, require further preparation. Preparation steps include: drying degassing filtering Both the eluent and the sample must be filtered for several reasons: 1. particles can prevent a check valve from sealing properly 2. particles can scratch piston seals causing them to leak 3. particles will accumulate on the column frit and plug it There are two methods for external filtration: 1. Solvent filter/degassers 2. Disposable syringe filters Solvent Filter/Degassers Because of the glass construction, Agilent degassers assure you of minimum contamination from contact with plastics. Additional benefits of using the degassers include: degas eluents as particulates are removed eliminate spurious peaks caused by outgassing in detectors eliminate pump downtime caused by air locks and particulates in check valve decrease piston wear; increase column life filter safely into plastic-coated or heavy-walled solvent reservoirs take all standard 47 mm filter membranes. Disposable Syringe Filters Syringe filters are convenient for filtering the sample. Agilent offers a variety of different membrane types and pore sizes. The filters are all based on cellulose materials that because of carefully controlled pore sizes offer good flow rates with minimal flow resistance. In-line Filtration

For in-line method filtration, the Agilent HPLC instrument provides intake and outlet filters installed within the system to trap wear particles.

9 For in-line method filtration, the Agilent HPLC instrument provides intake and outlet filters installed within the system to trap wear particles. The three types of filters within the Agilent 1 HPLC system are the inlet filter, the inlet valve and the outlet filter. It is important to change all filters on a regular basis to prevent instrument malfunctions. Description Agilent Part No. Inlet Filter Solvent inlet filter Inlet Valves Piston seal (2/pk) Sapphire plunger Outlet Filter PTFE frits (/pk) Should I just select the Bonus-RP column for all my method development with basic compounds? Basic compounds are best analyzed following the method development scheme outlined earlier in this brochure. Select an SB-C8 or SB-C18 column for initial development and use a buffered low ph mobile phase. Many times this approach provides a good separation and the StableBond columns will have exceptional lifetime at low ph even at high temperatures.

10 I m currently using a C18 column and my separation has a couple of peaks that elute early and a couple of peaks that elute late. What can I do to reduce the analysis time and maintain resolution of the early eluting peaks? First, you could try a gradient elution method on the C18 column. But many people do not like to use gradients, so choosing a different bonded-phase may help. Short chain polar bonded-phases such as the SB-CN and SB-Phenyl are ideal for separations like this. The increased polarity of these phases reduces retention of the later eluting hydrophobic compounds while often maintaining the retention of earlier eluting hydrophilic compounds. Using the same mobile phase conditions these three compounds are well resolved on the SB-CN in about minutes. The same separation on the SB-C8 column takes nearly twice as long and provides incomplete resolution. Can I really effectively use these very short columns on my HPLC instrument? Yes, you can. For columns of 3.0 mm i.d. and above, no instrument adjustments are necessary. For gradient separations with 2.1 mm and 1.0 mm i.d. columns the ideal HPLC is a high-pressure mixing instrument like the Agilent 1/1200 HPLC with the binary pump, because it minimizes the gradient delay volume. Using a low volume mixer and the injector by-pass (or micro-injector) further minimize gradient delay volume. Narrow i.d. tubing and a low volume detector flow cell are preferred but not necessary. These changes are easy to make and allow you to effectively use columns as small as the 2.1 x 30 mm columns or even the 2.1 x 1 mm columns. I'm working with environmental samples that may contain sulfur. I suspect that a few samples insufficiently cleaned up. Column performance has declined significantly. Is there any way to restore performance? How to Regenerate Heavily Fouled Columns Regeneration Procedure for Reversed-Phase Columns Disconnect the column and reconnect it to the chromatograph with the flow through the column in the reversed direction. Flush out any salts/buffers with HPLC grade water. Pump 2 ml of water through the column at 1 ml/min. Flush column with 2 ml of isopropanol. Flush column with 2 ml of methylene chloride. Flush column with 2 ml of hexane. Flush column again with 2 ml of methylene chloride. Flush column again with 2 ml of isopropanol. Reconnect the column to the chromatograph with the flow in the proper direction. Flush the column with the mobile phase without the buffer, the re-introduce the buffer. Equilibrate the column with 2 to 0 ml of mobile phase. Inject a standard or a sample to see if performance is restored. Note: For some retained compounds that have fouled the column, dimethyl-formamide may be a better "cleaning" solvent than methylene chloride and hexane. Regeneration Procedure for Normal-Phase Columns Connect the column to the chromatograph with the flow in the reversed direction. Flush column with 0 ml of 0:0 methanol:chloroform. Flush column with 0 ml of ethyl acetate. Reconnect the column in the proper flow direction. Equilibrate the column with 0 ml of mobile phase. Inject a standard or a sample and evaluate performance. Caution: In general, it is worth trying to clean and back flush an HPLC column with appropriate solvents before trying to replace the frit. There is always a danger that small amounts of packing material may be lost when a frit is replaced and column efficiency will decrease. In addition, the use of a 0. m in-line filter to capture particulate material and routine column washes with an appropriate strong solvent are highly recommended.

LC Column Troubleshooting. Do you have an equivalent column for my LC column?

LC Column Troubleshooting. Do you have an equivalent column for my LC column? LC Column Troubleshooting Do you have an equivalent column for my LC column? LC columns from different suppliers may have very different retention properties even if the bonding is the same. Although there