1 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 are some reference tables for columns from different suppliers, the result may change under different chromatographic conditions. We normally recommend an alternative column based on your application. Before we can recommend an alternative column, we need to know: 1.details of your analysis (sample composition and components to be analyzed; 2.if you can accept change in your LC conditions; 3.can you do some method modifications. If you cannot accept changes in the analytical conditions and/or the retention time, you should stay with your current column How do I select a guard column for an LC analysis? The function of a guard column is to protect the analytical column from the damage caused by particulate matter and strongly adsorbed material. To maintain an adequate capacity for sample impurities without introducing excessive peak dispersion, the volume ratio of the guard column to that of the analytical column should be in the range 1:15 to 1:25. Ideally the packing of the guard column should be the same as the analytical column so that the chromatography of the analytical column is not altered. Sometimes a similar packing may be used for the guard column. In our catalog, the analytical columns and their suitable guard columns are listed together. What is the effect on an LC separation of changing the particle size and column length? If particle size halves theoretical plates doubles (assuming column length remains the same). If particle size halves back pressure increases by a factor of four. If column length doubles theoretical plates doubles. If column column length doubles, analysis time doubles. As column length increases back pressure increases linearly. Here is an example of applying this information in column selection: A 200 mm long column packed with 10um particles generates about 6000 theoretical plates, an order of column efficiency that provides adequate separation in many cases. By reducing the particle size from 10um to 5um the efficiency of the same 200-mm column is doubled to about theoretical plates. However, this column generates a back pressure that is four times that of the one filled with 10um particles. Very often an efficiency of plates is not required, so the column length can be halved to 100mm, resulting in an efficiency of 6000 plates, and at the same time halving analysis time; the back pressure is only a factor two greater than the 200mm column with 10um particles. What is the effect on an LC analysis of changing the column diameter? If column diameter halves, sensitivity increaces by factor of four to five (assuming the injection volume is kept constant). For example,when a sample is injected onto a 2.1mm id column, the peaks are about a factor five higher than when the same amount of sample is injected onto a 4.6 mm id column. As long as the linear flow is maintained, parameters such as column efficiency, theoretical plates, back pressure and analysis time are unaffected by column ID reduction. What is the effect on the LC separation of changing the pore size? Smaller pore size allows higher linear velocity of mobiler phase. The optimum mobile phase linear velocity is determined by the particle size of the stationary phase. At the optimum linear velocity, the column generates the maximum number of theoretical plates. For columns with 4.6mm id, plate numbers are maximized at about 0.75 ml/min for 10um particles or 1.5 ml/min for 5um particles.
2 What can cause a loss in column efficiency (theoretical plates)? Column related causes: Cause Action Column developed a void Fill in the void with similar packing material and reverse or replace the column "Worn-out" guard column Replace guard column Other possible causes: Excessive extra-column volume, injected sample volume too large, strength of sample solvent stronger than mobile phase, changes in mobile phase composition. What can cause ''mystery peaks'' in my LC analysis? Column related causes: Cause Action Column fouling Clean or replace column ''Worn-out'' guard column Replace guard column Non column related causes: Impurities in the mobile phase, additional components in the sample, air bubbles in the system, electronic problems. How do I prevent peak tailing and splitting? Column related causes: Cause Action Column fouling Clean or replace column Particulates on column Reverse flush column Replace inlet frit Column void or channel Replace column or fill void "Worn-out" guard column Replace guard column Bad sample distribution Change to a column with a better inlet design Non column related causes: Excessive extra-column volume), sample overload, changes in mobile phase composition (additives, ph, buffer concentration). How do I buy columns from additional lots to do my method validation testing? First, find the lot number for your column(s) by checking the outside of the box in which it was shipped. The lot number is in small print, usually underneath the bar code. The lot number typically starts with the letter "B" followed by five digits. For example, lot number B99091 is used in the example below. If you no longer have the box, look at the tag on the column for the serial number. To determine the lot number from the serial number, call Agilent HPLC Column Technical Support at They will ask the Agilent manufacturing division to look up the lot number, and determine which other lots are available. Once you have the lot numbers you need,. or know your column lot number, use the following procedure for ordering columns: 1. Initiate a purchase order through Agilent Technologies or through your preferred distributor. 2. Use the special part number, , which signifies that the order is a special request..
3 3. Describe the desired column in terms of its type, dimensions and particle size. Include the normal part number for the column as part of the description. 4. Designate which lot(s) you want the column to be packed from. Alternatively, specify which lot(s) from which the column should not be packed. Example Description Part Number Eclipse XDB-C18 analytical column, 4.6 mm x 250 mm, ( ) Special Request for lots other than B99091 and B99079 Columns to be used for method validation purposes Which guard columns and guard hardware should I use for ZORBAX standard cartridge columns and all Agilent cartridge columns? Answer: ZORBAX Columns with ZORBAX Hardware Except in special circumstances, you should try to match exactly the guard column with the same packing and internal diameter as the column you're trying to protect. Find the part number and description of the column you have (or are ordering) on the Agilent web site or in our catalog or column selection guide. Find the part number and description for a guard column with the same (or less retentive) packing material. Select the part number for the appropriate guard column, based on whether the column is an analytical (4.6 mm, 4.0 mm, 3.0 mm Solvent Saver), narrow-bore (2.1 mm), semi-preparative (9.4 mm) or preparative (21.2 mm) column. For example, if your column is an analytical column, 4.6 mm x 150 mm, 3.5-micron Eclipse XDB-C8, you should select an XDB-C8 analytical guard cartridge, with dimensions 4.6 mm x 12.5 mm. If you're using a 2.1-mm i.d. narrow-bore column, select a narrow-bore 2.1 x 12.5 mm guard column. Both of these guard columns use the same hardware kit ( ). ZORBAX PrepHT columns (21.2 mm i.d.) have their own integral guard columns and have hardware for the inlet end for use of a guard cartridge and the end fittings. See Table 1 for several specific examples. Table 1 Guard Column and Hardware Selection Column Description Eclipse XDB-C8 4.6 x 150 mm, 3.5-um analytical column Eclipse XDB-C8 2.1 x 150 mm, 5-um narrow-bore column StableBond SB-C x 250 mm, 7- um PrepHT column Column Part Number Guard Column Description Analytical 4.6 x 12.5 mm 5-um Narrow-bore 2.1 x 12.5 mm 5- um PrepHT guard column 17 x 7.5 mm 5-um Guard Part Number Guard Hardware Part Number (guard hardware) (end fittings) Columns Having the Agilent Designation For ZORBAX and other manufacturers' columns that are sold in an Agilent cartridge format, catalog entries are designated by 'AC' in white letters within a black diamond. For Agilent cartridge columns you must purchase the guard cartridges separately in the given packing material. But the guard cartridge holder is an integral part of the Agilent cartridge column hardware for 2, 3, 4 and 4.6 mm i.d. configurations. The cartridge hardware kit consists of two end fittings and two sets of stainless steel collets. When the cartridge column is used without a guard column, the inlet end collets are installed in one direction; when the cartridge column is used with a guard column, the collets are installed in the reverse direction to allow the 4 x 4 mm guard cartridge to fit within the inlet column cartridge holder. Replacement filters may be used to protect the cartridge column from particulates (part no for 4 and 4.6 mm cartridges, part no for 2 mm cartridges), but are not required. A mounting tool (part no ) is also needed for installing the replacement filters, and is included in the cartridge holder kit (part no ). What is the difference between the StableBond SB-C8 and Rx-C8 columns? The SB-C18 and Rx-C18 columns? There is no difference between StableBond SB-C8 and Rx-C8. These columns are identical. The Rx-C8 column was the first column to use the sterically protecting bonding on the ultra-pure, fully hydroxylated Rx-silica. This bonding provides exceptional column lifetime and good peak shape at low ph. The bonding reagent is a diisopropyloctylsilane. The diisopropyl groups are the bulky side groups responsible for the steric protection of the bonded-phase against acid hydrolysis. A complete family of sterically protecting bonded-phases was then developed, including the SB-C18, SB-C8, SB-Phenyl, SB-CN and SB-C3. These are known as the StableBond family of columns. For consistency, the SB-C8 was added to the family, but the Rx-C8 was not dropped for the convenience of those currently using the Rx-C8 column.
4 What causes peak tailing in reversed-phase HPLC and what can I do about it? Some type of secondary interaction between an analyte and the column causes peak tailing. This interaction is in addition to the partitioning behavior seen for reversed-phase analyses. Peak tailing is most commonly seen with basic compounds and is usually a result of interactions between the residual silanols and positively charged basic compounds. The most common of these interactions is an ion exchange interaction between a positively charged basic compound and a negatively charged column surface silanol. Silanols on the surface of silica-based columns will have a negative charge when the ph of the mobile phase is above Therefore the quickest way to reduce peak tailing is to operate with a buffered mobile phase at a ph below 4. Choosing newer columns with high purity, fully hydroxylated silica will also minimize peak tailing because silanol activity and ionization is reduced. Some ZORBAX columns that use this type of silica are the StableBond columns, the Eclipse XDB columns, the Bonus-RP and the Extend-C18 column. Each of these columns can reduce peak tailing, but they are all a little different. The StableBond (SB) columns are ideal at low ph so they are often first choices to reduce peak tailing when using a low ph mobile phase. The Eclipse XDB columns are the first choice to reduce peak tailing if your mobile phase is ph 5 9. This column is double endcapped so it minimizes peak tailing by covering as many residual silanols on the column surface as possible and eliminating possible secondary interactions with silanols. The Bonus-RP column is also a good choice to reduce peak tailing in this intermediate ph region. The Bonus-RP column has a bonded-phase with an imbedded polar group. This group reduces interactions between basic compounds and residual silanols thereby improving the peak shape of basic compounds. This column can be used from ph 2 8. The Extend-C18 is designed as a high ph column and can be used up to ph At high ph many basic compounds are no longer charged and interactions with silanols are minimized, reducing peak tailing. Careful choice of a mobile phase can also reduce peak tailing. Buffered mobile phases (25 50 mm) will reduce peak tailing and low ph mobile phases are preferred (ph 2 3). This should also result in more reproducible chromatography. Mobile phase additives such as triethylamine (TEA) can be added to reduce peak tailing of basic compounds, if needed. TEA acts as a competing base and ties up silanol sites, eliminating interactions between your analyte and residual silanols. But this type of additive is rarely needed at low ph and is only occasionally necessary at intermediate ph. If you have peak tailing with an acidic compound the same process applies. Reduce the mobile phase ph to try to protonate the acids, then use a buffered mobile phase and try increasing the ionic strength of the mobile phase. Finally a competing organic acid can be added to the mobile phase and we have achieved excellent results with 0.1% trifluoroacetic acid (TFA), and this additive has a very low UV cutoff. Following these suggestions should reduce peak tailing of acids and bases. Most columns now use spherical particles because columns packed with spherical particles will have higher efficiencies. Therefore start by choosing a column with spherical particles. The most common particle size choice for analytical separations is 5 um because it is easy to use, but more often today the better choice is 3.5 um particles. These smaller particles generate higher efficiencies in shorter column lengths and make it possible to do separations with shorter analysis times. If analysis time is important to you, consider choosing a ZORBAX Rapid Resolution (3.5 um) column to minimize analysis time. The 4.6 x 150 mm, 3.5 um Rapid Resolution column will have the same efficiency as a 4.6 x 250 mm, 5 um column and reduce analysis time by 40%. Other shorter Rapid Resolution columns (75 mm, 50 mm, 30 mm, and 15 mm) are available to further reduce analysis time. Column pore size is selected based on the molecular weights of your analytes. A pore size of less than 100Å can be used for small molecules with molecular weights less than Larger molecules, such as proteins and peptides, should be analyzed on 300Å pore size columns. In addition, some smaller molecules with large, multi-ring, rigid structures can better be analyzed on 300Å pore size columns. Choosing the right pore size is important because most of the bonded-phase resides in the pores of the particles, therefore optimum retention and peak width are achieved only if the molecules can diffuse in and out of the pores rapidly and easily. How do I know whether I need a guard column or not? A guard column acts as a trap for many particulates and strongly retained components arising from samples and your HPLC system itself, and is installed immediately before the analytical column. The use of a guard column is a cost-effective way to increase substantially the lifetime of your analytical column (sometimes by up to 3-fold). Guard columns are recommended especially when biological or environmental samples are analyzed, and can also be very beneficial when analyzing pharmaceutical and agrochemical active ingredients, in-process samples, and formulated samples. In order to get the maximum benefit from the guard column, it is important to change it before its capacity has been exceeded and contaminants have reached the analytical column. Some guidance on how often to change guard columns is also available. A guard column, however, is not a substitute for a precolumn filter, which is always strongly recommended to protect the column(s) from particulates from samples, pump seals, and injector valve wear. A precolumn in-line filter should be placed immediately before the guard column (or analytical column, if no guard column is used). Agilent sells a very low dead volume in-line filter, which can even be used before Rapid Resolution HT 1.8 micron columns, with very little loss in performance.
5 How can I determine when to change my guard column? Many chromatographers change guard columns after a certain number of samples or standards and samples have been analyzed. This sample number is usually determined experimentally due to earlier column failure from waiting too long to change the guard column or by increasing the number of analyses before guard column change from a more conservative initial value. This guard column change interval is usually determined empirically for each method/sample type combination, but many analysts change guard columns either based on a finite number of sample runs or based on some column performance criterion such as a 10% increase in peak width or tailing factor, or a 10% decrease in column efficiency (plates). What is HILIC? HILIC stands for Hydrophilic Interaction Chromatography and is a term introduced in 1990 to distinguish it from standard normal-phase chromatography (Alpert J.Chromatography, 499 (1990) ) HILIC is a variation of normal-phase chromatography It offers greater retention than reversed-phase columns for very polar base The stationary phase is a POLAR material like as silica, cyano, amino, diol, etc. The mobile phase is organic with small amount of aqueous/polar solvent The polar solvent(s) (like Acetonitrile, alcohols, water) provide the strong eluting power How Does HILIC Work? HILIC Mechanisms on Silica The polar analyte partitions into and out of adsorbed water layer Charged polar analyte can undergo cation exchange with charged silanol groups (ph dependent ) Combination of these mechanisms results in enhanced polar retention Lack of either of these mechanisms results in no polar retention Agilent standard Zorbax columns can be used for HILIC applications if the shipping solvent is exchanged from the non-polar Heptane to a more polar solvent that is used for the HILIC application. Possible Zorbax columns are: Rx-SIL SB-CN, NH2 Please refer attached file on Hydrophilic Interaction Chromatography (HILIC) Separation of Basic Drugs using MS/MS Detection. How do you evaluate for column voids? First check your method and find out if you have operated at a ph higher than is recommended for the column. This is probably the major cause of column voids because silica can dissolve causing the column void. The sample injection solvent as well as the mobile phase needs to be considered here. If there is a column void, you will see a change in peak shape - tailing, broadening, or split peaks - on every peak in the chromatogram. A void will not usually cause a change in only one peak in the chromatogram. It also does not typically cause a change in analyte retention. You can also turn the column around and if there is a column void then the peak shape should be poor in the reverse direction as well. The only definitive way to check for a column void is to open the column and this should be done only as a last resort in identifying the problem with the column. What solvents do you recommend to clean the columns? I use THF. It is recommended to use the organic solvent acetonitrile to clean reversed-phase, C8 or C18, silica-based HPLC columns. THF is a strong reversed-phase organic solvent and that may be difficult to remove from hydrophobic bonded-phases, causing chromatographic variability. For most situations, acetonitrile is a strong enough solvent to remove sample and mobile phase components that have accumulated on your column during routine isocratic use. Before cleaning the column with organic solvent however, be sure that you have removed any buffer salts that may be in your mobile phase and column to avoid precipitation of these salts in the column. To do that, prepare your mobile phase without buffer salts and flush your column with column volumes of this mixture. That is, if your mobile phase is fifty-percent acetonitrile and fifty-percent 50 mm phosphate buffer, then the appropriate first step would be to remove the buffer salts with 50/50 mixture of acetonitrile and water. Then I would suggest flushing the column with pure acetonitrile thoroughly for column volumes. If you believe there may particulates at the top of the column, e.g., pressure may be higher than normal, you should consider disconnecting the column from the detector and reversing the direction of flow before the cleaning step. Do no reconnect the column to the detector, but allow the cleaning solvent, in this case, acetonitrile, to collect in a beaker. Since acetonitrile is commonly recommended for long-term column storage, the column can be easily stored after cleaning using the procedure described above. When ready to re-use this stored solvent, remember to flush the column with your mobile
6 phase composition without buffer salts before introducing the buffered mobile phase, again to reduce the possibility of salt precipitation. If one peak in a chromatogram is tailing but the others are not, what is the likely cause? Since I don't know the chemistry of the sample or details regarding the mobile phase or the column it is difficult to answer this question. Peak tailing can be caused by a variety of reasons and I would prefer to ask you several questions about your sample before I submit a response. Since most of the peaks in your chromatogram are well shaped and only one is tailing, I suspect that the chemistry of the column and sample are such that a secondary interaction is inducing the tailing. Modifying the mobile phase or selecting another HPLC column can reduce these secondary interactions. Rapid resolution columns are more likely to fail than 5 µm columns. Is it true? No, they are not. The Rapid Resolution columns are as rugged as the 5 µm, 250 mm columns. There are two types of accelerated column failure attributed to using smaller particles. First, smaller particle columns are thought to plug faster. This is not true when a standard 2 µm frit is used at the top of the column. Because of careful particle size control and the use of 3.5 µm particles, ZORBAX Rapid Resolution columns will not contain any particles as small as 2 µm. This means a standard frit can be used on the column and the Rapid Resolution columns will be no more prone to plug than a 5 µm particle size column. Second, columns with smaller particles are thought to have shorter lifetimes because the column beds compress, leaving voids that cause peak broadening and tailing. It is true that 3.5 µm particle size columns will operate at slightly higher pressures than 5 µm columns, but ZORBAX particles can easily withstand these increases in pressure. ZORBAX particles are packed at 8000 psi and can easily withstand pressures up to 5000 psi in routine use. A Rapid Resolution 4.6 x 150, 3.5 µm column will typically be operated below 3000 psi, so the column bed will not compress when using ZORBAX Rapid Resolution columns. So both the rugged ZORBAX particles and the standard 2 µm column frit assure you long column lifetime when using Rapid Resolution columns. The very short Rapid Resolution columns can also be used at high flow rates to further reduce analysis time. What do you think about neutralizing extra-column volume by packing short columns with wide-bores (7mm)? As long as you are willing to accept operating at a higher flow rate. If you operate at 1.0 ml/min on a 4.6 mm i.d. column, then operating at 2.3 ml/min would be the equivalent linear velocity when using a 7.0 mm i.d. column. Solvent waste may be an issue. Can I use some detergents for cleaning my HPLC columns? Detergents are not recommended for cleaning reversed-phase (RP) HPLC columns. Ionic detergents are typically long-chain carbon compounds having an ionizable group at one end and have been used as ion-pairing agents. Because these long carbon chains partition well into the bonded phase of reversed-phase columns (i.e. the bonded-phase of the C18 reversed-phase - column strongly retains the long carbon chain of a detergent), the removal of detergents can be difficult, if not impossible. Some manufacturers? indicate (newer) columns can be reversed to remove blockage from the front of the column. Is this generally recommended or does it depend on the column? HPLC columns are more efficient and packed better than years ago, therefore this is generally recommended for most reversedphase and normal phase silica based columns. This procedure is designed to remove particles from the column frit when high pressure occurs at the column inlet, but it will not work all the time. Because it does not require opening the column it is worth trying. Make sure when you do this that you disconnect the column from the detector and make sure the particles that plugged the column are not coming from the HPLC system or you may just plug the frit at the back end of the column and this may not be replaceable. Columns can also be turned in the reverse direction for washing/cleaning with stronger solvents to remove adsorbed material. This has the benefit of not exposing the rest of the column to the contaminants. When this is done the column should also not be attached to the detector.
7 How do I clean my C4 HPLC column? Much as you would any RP-phase column and I've included some general instructions provided that you are using standard mobile phase conditions to separate small molecules. If you are separating peptides and proteins or if your sample is dissolved in plasma, then the guidance is different and it is recommend that you call Agilent technical support for more information or contact your local Agilent sales office. What is the purpose of the H3PO4 wash of the HPLC column? A phosphoric acid wash has been shown to be effective at reducing tailing caused by the sample complexing with metals in the HPLC system. Typically a 1% phosphoric acid wash of the system and column is suggested to eliminate this tailing and it works. It is perfectly reasonable to use these recommended wash conditions with Agilent ZORBAX StableBond reversed-phase HPLC products. The StableBond HPLC column is particularly stable at low ph - the SB-C18 column is stable at a ph of 0.8 and 90 C. How can you tell if your peak tailing is caused by metal complexation? Look to see if a lone pair of electrons on either a N or O atom can chelate with the metal to form a 5 or 6-membered ring. Metal complexation is a commonly overlooked cause of peak tailing and metals are present in every HPLC system. What is the recommended Regeneration Procedure for Reversed-Phase HPLC Columns? 1. Disconnect the column and reconnect it to the chromatograph with the flow through the column in the reversed direction. 2. Flush out any salts/buffers with HPLC grade water. Pump 25 ml of water through the column at 1 ml/min. 3. Flush column with 25 ml of isopropanol. 4. Flush column with 25 ml of methylene chloride. 5. Flush column with 25 ml of hexane. 6. Flush column again with 25 ml of methylene chloride. 7. Flush column again with 25 ml of isopropanol. 8. Reconnect the column to the chromatograph with the flow in the proper direction. Flush the column with the mobile phase without the buffer, then re-introduce the buffer. 9. Equilibrate the column with 25 to 50 ml of mobile phase. 10. 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. What is the recommended Regeneration Procedure for Normal-Phase HPLC Columns? 1. Connect the column to the chromatograph with the flow in the reversed direction. 2. Flush column with 50 ml of 50:50 methanol:chloroform. 3. Flush column with 50 ml of ethyl acetate. 4. Reconnect the column in the proper flow direction. 5. Equilibrate the column with 50 ml of mobile phase. 6. 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 0.5?m in-line filter to capture particulate material and routine column washes with an appropriate strong solvent are highly recommended.
8 How can I properly wash GPC HPLC columns (safely)? Wash the column in the reverse direction, not attached to the detector and at half the recommended flow rate (keep the pressure below the recommended maximum). First choose a solvent that will dissolve what you believe has contaminated the column. Most GPC columns are PS-DVB and you need to check the solvent compatibility before using a solvent. Many wash solvents are a higher viscosity than the typical eluting solvents so a lower flow rate with careful attention to pressure is needed. Anionic samples can adsorb onto PS-DVB and if these have contaminated your GPC column a wash solution with a salt is recommended. Check to see what types of salts are recommended for the column. In some cases the polarity of the material adsorbed may require washing with organic solvents modified with acid (formic or acetic) or base (triethanolamine) (check the ph range) or some water may be compatible with an appropriate organic solvent. If more hydrophobic material were retained, elevated temperature along with an appropriate organic solvent would be recommended. Once again you need to check the maximum temperature range allowed for your column. If you wash carefully the column should not degrade from the solvent switching. Is there any kind of 'generic' column test to track the performance of a HPLC column? How often should it be completed? In my opinion, the best way to evaluate a columns performance is to use the QC test that should be shipped with each HPLC column provided to you. By comparing the efficiency, retention and peak shape of the peaks in the sample and very importantly the pressure under these experimental conditions, you will be able to tell if your column has changed over time. These tests can also tell you how your column has changed, as these are the same tools we use to diagnose a column problem. Significant changes in retention, can suggest loss in bonded phase and significant changes in peak width/efficiency and pressure can suggest column contamination. Dramatic losses in peak width and efficiency suggest a column void. Most manufacturers use similar solutes and mobile phase test conditions to QC their columns. However, different bonded phase require modification of the amount of organic modifier to achieve reasonable retention. What's important is that that you QC-test a column on your system before you use it for a particular project. Then you know how that column performs on your system, before you start injecting samples and before you start having problems. If problems develop, use this QC-test to verify that the HPLC system and the column are performing well. Before you retire a column from a project, clean and QC-test the column. If for any reason, you start a project with a used column, at a minimum, QC-test the column before injecting samples. What types of column are dissolved above ph 7? Many commercially available silica-based HPLC packings dissolve at ph 7, for the simple reason that silica is soluble at ph 7. Therefore, Agilent has developed technology to significantly reduce silica dissolution and extend the usable ph range of silicabased columns. The ZORBAX Extend-C18 can be effectively used above ph 8 and even up to ph Bidentate-bonding is key to this enhanced stability while offering the high efficiency that only silica can provide. How do I make my ZORBAX RRHT LC columns last even longer? Rapid Resolution Liquid Chromatography offers dramatic time savings and increased resolution thanks to the column s very small 1.8 µm particle size, which needs inlet frits of only 0.2 µm pore size. The drawback to this smaller pore size is that the smallest particles contained in the mobile phase or any wear debris from the pump, the autosampler switching valve or other contamination are retained by the column s inlet frit. With a pore size that small, the columns get blocked easily, because the frits get plugged very quickly. The best and most efficient way to prevent this and extend column life is to use an in-line filter in front of the column. Agilent now offers two new in-line filters, which work at pressures up to 600 bar. One filter has a 0.2 µm pore size frit with 2.1 mm diameter for use with 2.1 mm ID ZORBAX RRHT LC column. A low volume taper ensures that the liquid flow is always evenly distributed for efficient use of the full filtering capacity of the frits. The low volume of this 2.1 mm filter prevents extra external band spreading and preserves the high efficiency of the ZORBAX RRHT LC column. Figure 1. RRLC In-line filter.
9 A second filter comes with 4.6 mm diameter frits and 0.2 µm pore size for use with 3 and 4.6 mm ID RRLC columns. Every kit comes with 3 frits and one connecting capillary. Disposable replacement frits are also available in conveniently sized packages. The same design offering similar benefits is also used on our 400 bar in-line filters. Figure 2: 400 bar In-line filters. Agilent also offers new fingertight polyketone fittings for use up to 600 bar. Fingertight PEEK fittings can only be used up to 400 bar, but these new fittings allow easy column exchange and seal tightly up to 600 bar and 100 C to offer the best alternative for high pressure RRLC applications. Ordering information for filters and fittings follows, and you can learn more by reading Rapid Resolution HT Columns Maintain High Resolutions with Agilent In-line (Pre-Column) Filters. Figure 3: New fingertight polyketone fitting for use up to 600 bar.
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The Role of Pore Size in Reversed Phase HPLC SGE is excited to launch a new HPLC product line under the ProteCol brand. Fundamental to the new ProteCol line of columns is the continued focus on inert column
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CONTENTS I. INTRODUCTION II. II. GETTING STARTED a. Column Connectors b. Column Installation c. Column Equilibration d. ecord Installation e. Initial Column Efficiency Determination f. VanGuard Pre-columns
CAPCELL PAK C18 IF2 Type http://hplc.shiseido.co.jp/e/column/html/if2_index.htm Page 1 of 2 List of Sales Representatives Contact Shiseido Technical Materials Catalog List HPLC Columns HPLC Instruments
Acclaim Mixed-Mode WCX-1 Product Manual for the Acclaim Mixed-Mode WCX-1 Column Page 1 of 28 PRODUCT MANUAL for the Acclaim Mixed-Mode WCX-1 Columns 4.6 x 150 mm, P/N (068353) 4.6 x 250 mm, P/N (068352)
William E. Barber, Ph.D. Applications Chemist ctober 0 00 Secrets of Good Peak Shape in HPLC Time: :00 p.m. CET Telephone Number: + 44 0 76 088 Chairperson: John Vis The Secrets of Good Peak Shape in HPLC
Guide to Choosing and Using Polymer Reversed Phase Columns! Choosing the best RP Column to use: Silica is normally used as the packing material for HPLC columns for a number of reasons. It is very strong,
Chemistry 4631 Instrumental Analysis Lecture 28 High Performance Liquid Chromatography () Instrumentation Normal Phase Chromatography Normal Phase - a polar stationary phase with a less polar mobile phase.
HPLC Columns Shodex TM ODP2 HP series columns Better retention of highly polar substances Technical notebook No. 6 Contents 1. Introduction 1-1. Specifications 1-2. Eluent Compatibility of ODP2 HP Series
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1 Reverse Phase How proteins separate on reverse-phase HPLC RP chromatography separates proteins through the interaction of the hydrophobic foot of the protein with a nonpolar surface of the particle RP
Primesep 5 µm columns Primesep columns feature double functionality of the bonding i.e : alkyl chain with anionic or cationic group, chelating group. This feature creates unique selectivities when using
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LEARNING OBJECTIVES CHEM 212: SEPARATION SCIENCE CHROMATOGRAPHY UNIT Thomas Wenzel, Bates College In-class Problem Set Extraction Problem #1 1. Devise a scheme to be able to isolate organic acids, bases
"Theory and Practice of High Speed Chromatography for Bioanalysis" Stuart Coleman March 20, 2007 General Observations about HPLC Separations A good separation is necessary for good bioanalytical quantitation.
Welcome to our E-Sear: Choosing HPLC Columns for Faster Analysis Smaller and Faster High Throughput/Fast LC Requires. Short columns 0 mm or shorter Small particle sizes. µm Rapid Resolution or new.8 µm
Part 1. General Chromatographic Theory Part 2. verview of HPLC Media Part 3. The Role of the Mobile Phase in Selectivity Part 4. Column Care and Use Reversed Phase Solvents 2 Solvents for RP Chromatography
HPLC Separation Fundamentals Ed Kim Application Engineer Agilent Technologies, Inc January 14, 2009 Separation fundamentals Presentation Outline Major HPLC modes Key Equations Resolution van Deemter Common
User Manual Acclaim Mixed-Mode WAX- Columns 06565 Revision 03 October 05 Product Manual for the Acclaim Mixed-Mode WAX- Column Page of 3 Product Manual for Acclaim Mixed-Mode WAX- Columns 5µm, 0 x 50 mm,
June 009 ewsletter Pharmaceutical Analysis: What is Your Problem? SIELC Technologies, Inc., Prospect Heights, IL 0070 Pharmaceutical analysis involves liquid chromatography of various compounds, from active
Pure Chromatography Consumables Pure flexibility. Pure specialization. Pure convenience. Pure Consumables More focus on your application The Pure consumable portfolio offers an unrivaled range of products
New 5-micron HALO-5 columns based on Fused- Core particle technology boost the performance of HPLC. Compared to other HPLC columns, HALO-5 columns have: the highest plate number versus any other 5-micron
HPLC Columns MANUAL HILICpak VT-50 2D Shodex HPLC Columns Europe, Middle East, Africa, Russia For technical support please use contact details shown below: SHOWA DENKO EUROPE GmbH Shodex Business Konrad-Zuse-Platz
LC III: HPLC What is HPLC? Originally referred to as High-Pressure Liquid Chromatography Now more commonly called High Performance Liquid Chromatography In general: The instrument controlled version of
Technical Note 101 Method Development in Solid Phase Extraction using Non-Polar ISOLUTE SPE Columns for the Extraction of Aqueous Samples This technical note includes by specific information on the extraction
Page 1 of 13 CIPAC/4105/R GUIDELINES FOR THE DESIGN OF CHROMATOGRAPHIC ANALYTICAL METHODS INTENDED FOR CIPAC COLLABORATIVE STUDY Prepared for CIPAC by Dr M J Tandy*, P M Clarke and B White (UK) The rapid
High Pressure/Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the
User Manual IonPac NS2 Columns 065501 Revision 02 October 2015 For Research Use Only. Not for use in diagnostic procedures. Product Manual for IonPac NS2 Columns IonPac NS2, 5µm, 4x250mm Analytical Column
Ensure reproducible Polymeric SPE workflow for high-throughput bioanalysis applications Agilent Bond Elut Plexa vs. Waters Oasis Sample Prep Products Reliability is a key parameter in developing rugged,
HPLC Winter Webinars Part 2: Sample Preparation for HPLC Jon Bardsley, Application Chemist Thermo Fisher Scientific, Runcorn/UK The world leader in serving science What am I Going to Talk About? What do
Chem 230, Fall, 2014 Homework Set # 3 Short Answer SOLUTIONS 1. List two advantages of temperature programming in GC. a) Allows separation of solutes with widely varying retention factors in a reasonable
Chromatography Chromatography is essentially the separation of a mixture into its component parts for qualitative and quantitative analysis. The basis of separation is the partitioning of the analyte mixture
HPLC COLUMNS Theory, Technology, and Practice Uwe D. Neue with a contribution from M. Zoubair El Fallah WILEY-VCH New York Chichester Weinheim Brisbane Singapore Toronto CONTENTS Preface ix 1 Introduction
Peptide and protein analysis by capillary HPLC Optimization of chromatographic and instrument parameters Application Angelika Gratzfeld-Huesgen Abstract This application has been verified using an Agilent
CONTENTS I. INTRODUCTION II. III. GETTING STARTED a. Column Connectors b. Column Installation c. Column Equilibration d. ecord Installation e. Initial Column Efficiency Determination COLUMN USE a. Sample
columns Acclaim Mixed-Mode WCX- for Separating Basic Molecules The Acclaim Mixed-Mode WCX- is a novel, high-efficiency, silica-based column specially designed for separating various basic analytes. This
TG 0-0 Hypersil BDS Columns Introduction Hypersil BDS columns have gained a reputation over the years as one of the most robust, reproducible and reliable HPLC column brands available. This Technical Guide
L2 Page1 Instrumental Chemical Analysis Chromatography (General aspects of chromatography) Dr. Ahmad Najjar Philadelphia University Faculty of Pharmacy Department of Pharmaceutical Sciences 2 nd semester,
Unexpected Peaks in Chromatograms - Are They Related Compounds, System Peaks or Contaminations? From the Diary of an HPLC Detective SHULAMIT LEVIN HPLC in Pharmaceutics Σ Stability Indicating Methods Extra
The Theory of HPLC Ion Pair Chromatography i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.
XSelect High Strength Silica (HSS) hplc Columns Contents I. INTRODUCTION II. CONECTING THE COLUMN TO THE HPLC SYSTEM a. Column Connection b. Column Connectors and System Tubing Considerations c. Band Spreading
Potential sources of contamination on Mass Spectrometers and suggested cleaning procedures Purpose: This document provides information on potential sources of background contamination commonly encountered
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Gel Permeation Chromatography (GPC) or Size Exclusion Chromatography (SEC) Size Exclusion Chromatography (SEC) is a non-interaction based separation mechanism in which compounds are retained for different
Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks bjective: The purpose of this experiment is to quantify the caffeine content of a diet soda sample using Ultra-High Performance
P R O D U C T B U L L E T I N Fused-Core particle technology for hyper-fast and super-rugged HPLC columns HALO column packings are not made the typical way. Instead, the particles packed into HALO columns
HPLC Preparative Scaleup of Calcium Channel Blocker Pharmaceuticals Application Pharmaceuticals Author Cliff Woodward and Ronald Majors Agilent Technologies, Inc. 2850 Centerville Road Wilmington, DE 19808
COMPARISON GUIDE to C18 Reversed Phase HPLC Columns Comparison Data on 60 Commonly Used C18 Phases Stationary Phase Specifications Phases Compared According to Relative Hydrophobicity Phases Compared According
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Description of Module Subject Name Paper Name 12 Module Name/Title 13 1. Objectives 1.1. To understand the basic concept and principle of 1.2. To understand the components and techniques of 1.3. To know
A C18 lica Column With Exceptional Temperature and ph Stability Brian A. Jones 1, Stephanie J. Marin 1, Jody Clark 1, Nathan L. Porter 1, J. Andreas Lippert 2, and Todd M. Johnson 2 1. Selerity Technologies,