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The HPLC System Total Cost of Ownership Calculator helps you estimate your total cost for purchasing and running a Thermo Scientific HPLC system over a designated time period. Compare against other instruments to find the best product for you. Input your typical system use conditions and typical. In this first article, I will take you through the principle behind HPLC and remind you of its uses – you will be ready for the lab in no time! How does HPLC work? High-performance liquid chromatography, or HPLC, is a long name for a powerful technique based on the simple fact that individual compounds behave differently in water.
The MTS HPLC calculator is in the form of an Excel spreadsheet comprised of 7 worksheets which correspond to useful HPLC related calculations. The calculator has been downloaded hundreds of times by users all over the world.
If you have any requests or feedback then please.The calculations provided are as follows:Column EquilibrationHow long do you need to equilibrate a column for before it is ready to use?Void VolumeCalculates the void volume and estimates the retention time of t0.Pressure ConverterChange pressure units between the most commonly used units, e.g.
Reverse Phase HPLC Basics for LC/MSAn IonSource TutorialbyAndrew GuzzettaThis Tutorial was first published July22nd, 2001 We were going to call this tutorial 'Reverse PhaseHPLC for Proteomics' but wedecided to exercise some restraint. We decided to write this tutorial because reverse phase chromatography is themost common form of chromatography used in LC/MS applications. This tutorial is basically targetedto students and those that are new to reverse phase chromatography, HPLC, andLC/MS.The tutorial addresses RP HPLC of peptides and proteins but theprinciples described can be applied toward the chromatography of any compound.Table of Contents.The message of this tutorial is that reverse phase HPLC issimple.
Compounds stick to reversephase HPLC columns in high aqueous mobile phase and are eluted from RPHPLC columns with high organic mobile phase. In RP HPLCcompounds are separated based on their hydrophobic character. Peptides can be separated by running a linear gradient of the organicsolvent. I often tell my fellow researchers to run the 60/60gradient when chromatographing an unknown.
The 60/60 gradient meansthat the gradient starts at near 100% aqueous and ramps to 60% organicsolvent in 60 minutes. The majority of peptides (10 to 30 amino acidresidues in length) will elute by thetime the gradient reaches 30% organic. To learn some of the simpleprinciples of RP HPLC please read on.In most cases the HPLC you intend to use must be able to pump and mix two solvents.
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This can be accomplished with one pump and aproportioning valve or by using two separate pumps. Generally the pumpingconfiguration is an aspect of the instrumentation that is transparent tothe user. Reverse phase chromatography can also be performed in a purelyisocratic mode where the solvent conditions are held constant, this formof reverse phase chromatography can be carried out with a single pump.Isocratic methods are used most often in a QC environment in which a single analyte has been extensively characterizedand the compound is being run to confirm it's identity and to lookfor closely related degradation products. Ifyou do not own an HPLC here is ato HPLC vendors and accessory suppliers.Components and Specifications. column dimension (size). particle size and pore size.
stationary phase. Since columns are tubular, columndimensions usually take the following format, internal diameter Xlength (4.6mm X 250mm). As a mass spectroscopist you willencounter columns ranging in internal diameter from 0.050 to 4.6 mmor even larger if you are performing large scale preparativechromatography. For mass spectrometry a short reverse phase column will work nearly as well as alonger column and this is an important fact because shorter columns are generallycheaper and generate less back pressure. Why is less backpressure important? If a column runs at low pressure it allowsthe user more flexibility to adjust the flow rate.
Sometimesshorter columns are used to do fast chromatography at higher thannormal flow rates. Interms of length weroutinely run 100 mm columns, however 50 mm or 30 mm columns may beadequatefor many LC/MS separation needs. The most common columns are packed with silica particles. The beads or particles are generally characterized by particleand pore size. Particle sizes generally range between 3 and 50 microns, with 5 um particles being the mostpopular for peptides. Larger particles will generate less system pressureand smaller particles will generate more pressure. The smallerparticles generally give higher separation efficiencies.
Theparticle pore size is measured in angstroms and generally range between 100-1000angstroms. 300 angstroms is the most popular pore size forproteins and peptides and 100 angstroms is the most common forsmall molecules. Silica is the most common particlematerial. Since silica dissolves at high pH it is not recommendedto use solvents that exceed pH 7. However, recently some manufactureshave introduced silica based technology that is more resistant tohigh pH, it is important to take note of the manufactures suggesteduse recommendations.
In addition the combination of hightemperature and extremes of pH can be especially damaging to silica. The stationary phase is generally made up of hydrophobic alkyl chains ( -CH 2-CH 2-CH 2-CH 3) that interact with the analyte. There are three common chainlengths, C4, C8, and C18. C4 is generally used for proteins and C18is generally used to capture peptides or small molecules. Theidea here is that the larger protein molecule will likely have more hydrophobicmoieties to interact with the column and thus a shorter chain lengthis more appropriate. Peptides are smaller and need the morehydrophobic longer chain lengths to be captured, so C8 and C18 areused for peptides or small molecules. Here is an interestingnote: Observations have been made that C8 columns are actuallybetter for capturing smaller hydrophilic peptides, the theory hereis that the longer C18 chains lay down during the early aqueousperiod of the gradient and the more hydrophilic peptides are notcaptured.
We use C8 routinely for all peptide work and thisparticular alkyl chain length works equally well if not better thanC18 for all peptides.The reverse phase solvents are by convention installed on the HPLC channelsA and B. The A solvent by convention is the aqueous solvent(water) and the B solvent by convention is the organic solvent (acetonitrile,methanol, propanol).It is important to follow this convention since the terms A and B arecommonly used to refer to the aqueous and organic solvents respectively. TheAsolvent is generally HPLC grade water with 0.1% acid. The B solventis generally an HPLC grade organic solvent such as acetonitrile ormethanol with 0.1% acid. The acid is used to the improve thechromatographic peak shape and to provide a source of protons in reverse phaseLC/MS.
The acids most commonly used are formic acid, triflouroacetic acid,and acetic acid. A0.1% v/v solution is made by adding 1ml of acid per liter of solvent. Triflouroacetic acid hasbeen reported to suppress MS ionization and often mass spectroscopistslower the percentage of TFA to 0.05 or even 0.02% without significant loss inchromatographic efficiency. Some MS people add a small percentage ofheptafluorobutyric acid (HFBA,from ) to acetic acid solvents or low TFA containingsolvents to help improve peak shape.
Since modern mass spectrometersare very sensitive it is important not to use plastic pipette tips whenadding acid to the mobile phase, always use glass. In our work we use acetonitrile as ourorganic solvent.We have heard that the best electrospray solvent is 30% methanol, 35 mMacetic acid.
We commonly use this solvent system for ESI MSinfusion, but have found that acetic acid is an inferior acid forchromatographic peak shape. Our preferred HPLC grade water, acetonitrileand methanol is purchased from.
Our preferredTFAcomes in 1 ml ampoulesfrom from.Our Preferred Solvent System for ESI LC/MSA = HPLC grade Water, 0.1% formic acidB = HPLC grade Acetonitrile, 0.1% formic acidWhen chromatographing an unknown we normally use the followingsimple gradient to learn about the hydrophobic character of theunknown compound. The% A in the gradient described below isimplied.We call this the 60/60 gradient, because we runfrom near 0% B to 60% B in 60minutes. Through experience we have noted that 90% of all peptides will elute from a C18 reverse phasecolumn by 30% acetonitrile. There may be a few really hydrophobicpeptides that elute later that is why we take the gradient to 60% B.You may even want to run this gradient to 80% at least once to see if youare getting everything off of the column.
You may ask why don't we start thegradient at 0% B? As we talked about before, in 0% organic and in high aqueous, the veryhydrophobic, long C18 alkyl chains in an effort to get awayfrom the high aqueous environment mat down on the particle. When these alkyl chainsmat down they are inefficient at capturingthe analyte so chromatographers in the know start the gradient with somesmall% of organic, 2-5%.It is important to use the correct flow rate for your HPLCcolumn. Below is a table with standard flow rates for easyreference. If you are running a column with a different diameter thanthose shown in the table please review the page to learn how to calculate the appropriate flow ratefor your column.Thesample is normally reconstituted in the A solvent to maximize binding to thecolumn.The sample should not be dissolved in an organic solvent or it may not stickto the stationary phase. The sample should not be dissolved indetergent containing solutions. Some detergents may bindto reverse phase columns and modify them irreversibly.
In additiondetergents preferentially ionize in electrospray mass spectrometry and canobscure the detection or suppress the ionization of the analyte.Once you have a separation you may want to optimize it. You may wishto shallow out thegradient to improve the separation, or you may wish to shorten the run time.Taking the illustration above one can see that all of the peptides are outby 40 minutes. This does not mean that we can change this 80 min run intoa 40 min run, but there is room for improvement.
The first step in theoptimization is to determine the%B at which the last peak elutes. If you look at the blue gradientline you might guess that the last peak elutes near 40%B but this would be incorrect.All HPLC systems have a gradient delay. The gradient delay is the timebetween when the software tells the pumps to start pumping at a certainmobile phase composition and the time it takes for that solvent compositionto reach the column and have an effect. A good guess for a gradientdelay is 10 minutes. This would mean that our guess for the finalmobile phase composition for the 40 min peak would be approximately30%B.
To observe the gradient delay time look at the illustrationabove and observe that the baseline returns to the starting conditions at 70minutes and not at 60.1 minutes when our pumps have gone back to 2% B. One must take care to avoid having thelast peak elute on the 'equilibration cliff', (at 70 min. In thisexample). This can be avoided by ending the gradient at a% Bthat is slightly higher than that required by the last component.Based on the separation shown at the top of this section onecould rewrite the gradient to look like this:This would make the gradient shallower and possibly give a better peak separation. To shorten the run time one could rewrite the gradient to look like this:This last change would cut 30 min. From the analysis time. Shorteranalysis times are always better for work efficiency.
With everyminute you can cut from the HPLC method without sacrificing yourchromatographic goals you will be rewarded with better work efficiency. Withthis change the last peakwould most likely still elute at 40 minutes and the peptide separation wouldessentially remain the same as in the initial 60/60 analysis.The column must be equilibrated, re-equilibrated to theinitial high aqueous solvent composition before another analysis can beperformed. Normally this re-equilibration is stuck onto the end of thegradient.
How much equilibration time is enough? As a rule ofthumb we give 20 minutes. In reality it depends on the column length,flow rate and the hydrophobicity of your peptides. Some chromatographers use10 minutes as their standard equilibration time. Equilibration is allabout fitness of purpose. You should determine the the equilibrationtime experimentally, the criteria will be, does myanalyte really stick to the column and chromatograph appropriately andreproducibly with subsequent analyses. If you choose to do this part of the method development you will undoubtedlybe rewarded with improved chromatography and better cycle time.Yes.
Scientists are control freaks. If you cancontrol a variable, control it! Actually if you are performing automatedanalyses over a long period of time peak retention times can drift withchanging ambient temperature. It is common for many companies andinstitutions shut down the air conditioning at night to save money, whichcould result in shifting peak retention times due to dramatic changes inambient temperature.Many HPLCs provide the option to control column compartmenttemperature. If your HPLC does not have this capability a heatedcolumn jacket can be purchased from many suppliers.
The most commonrunning temperature is 40C, this places the column compartment wellabove even the most extreme ambient temperature fluctuations. Inaddition to maintaining constant temperature, temperature can be used toinfluence the chromatographic separation. No chromatographic study iscomplete without a temperature study. In our experience highertemperature is better, peaks will be sharper and elute earlier. It is nottoo uncommon to perform chromatography at 60 C and some daredevils even goto 80 C. Remember though that higher temperature will lead to ashorter column lifetime and some columns may not be able to tolerate60 C.
Consult the manufactures recommendations when experimentingwith high temperature. After your runs are complete for the day it isadvised that you turn off your column heater since high temperature leads tostationary phase deterioration.One observation is that if you start up a reverse phaseanalysis from a dead stop with a column that has perhaps been sitting inhigh aqueous conditions for up to 10 hours the analysis will giveirreproducible results. Conventional wisdom has it, you want to firstflush the column with the highest% organic of your method for at least 3column volumes and then bring it back to the equilibrating condition.This practice may have the advantage of getting you to standardequilibration conditions faster and it will also clean your column. A better alternative is to makethe first run a blank run (or 'preparation run') and then the next run can be your real analysis. We prefer the second optionbecause it should get you to the standard starting conditions moreaccurately. However, often, if we are in a hurry and thefirst option is quicker, well.We store our columns in 50/50 methanol/water without anyacid.
If you are using a salt, unlikely in LC/MS, wash your entiresystem, solvent bottles, HPLC, solvent lines, and column, into a non-saltcontaining solvent. Salt may precipitate out and plug your HPLCor column or may cause corrosion. Usually we flush with pure waterfirst then leave the system in 50/50 methanol: water. Some salts mayprecipitate out in high organic so an initial water wash is advised.The 50/50 methanol:water solution helps to stop bacterial growth which can muckup your system. Take care of your HPLC, it's the right thing to do!: Triflouroacetic acid, formic acid,heptaflouobutyric acid and acetic acid are all very caustic reagents.Acetonitrile, methanol, and propanol are harmful solvents Consult the material safety data sheets(MSDS) that come with these reagents and getthe permission of the safety officer at you company or institutionbefore performing these experiments. Always wear the appropriate safetyapparel; safety glasses, lab coat, and gloves.
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Use a fume hood whenappropriate. If you are not trained in laboratory safety you shouldnot attempt these procedures. Read our disclaimer, follow thelink at the bottom of this page. Copyright 2001-2017 IonSource, Allrights reserved. Last updated:Tuesday, January 19, 2016 02:49:16 PMvisitors.