one of the major components of the liquid chromatography is the pumping system so i'm going to go through the requirements for each hplc pumping system the first is generation of pressures of up to 6000 psi we need a pulse free output you don't want the pulsing because that will give you different not smooth peaks in your chromatograph flow rates ranging from 0.1 to 10 milliliters per minute depending on your needs flow control and flow reproducibility of 0.5 relative and corrosion resistant components obviously they're going to be exposed to liquids and different types of liquids and we don't want corrosion to occur as you have to replace them and it also could ultimately react with your samples there are three types of pumping systems commonly used the first one is the reciprocating pump and this one is shown on the right right here so it consists of a small chamber in which solvent is pumped by back and forth motion of a motor driven piston and so here you see the motor the piston and so it's going to move in a back and forth motion and then as a result the solvent comes up here and the solvent will be pumped in by the back and forth motion of that piston the advantages um you have small internal volumes and typically we're talking 35 to 400 microliter high pressure outputs so pressure is up to about 10 000 psi there's zero in there psi 10 000 psi you get a constant flow rate and ready adaptability to gradient flow disadvantage is that we do produce a pulsed flow with the back and forth motor journalism second type is going to be a displacement pump and this one consists of a large syringe-like chamber equipped with a plunger that is activated by a screw-driven mechanism the advantages to this is you it's the flow independent of viscosity and back pressure so your flow can be consistent no matter what type of sample you have or what type of back pressure you have and you get output pulse free so you don't have that pulsing issue you have the previous one disadvantage is it does have limited solvent capacity the third type is the pneumatic pump this is a mobile phase contained in a collapsible container house in a vessel pressurized by a compressed gas advantages is it is inexpensive and pulse free disadvantage is that you have limited solvent capacity and pressure output it's dependence of flow rate on the solvent viscosity and back pressure and it's not amenable to gradient flow so you are limited in type of flow you limited in depending on your viscosity of your solvent back pressure so this one has definitely a lot more disadvantages than advantages although the price inexpensive price limit does make it advantageous to a lot of people next is our sample injection system so our sample injection system limit this is your limitation in precision issue with this is that the limitation will come from the reproducibility of sample introduction so you load your sample use some type of syringe and that sample inside here has a loop and so you put it in it fills the loop the loop fills with whatever volume and the rest goes to the vent it has a section going to the column a section coming from the pump and so when it's closed like this it sits in the loop and waits when you're ready to inject the sample you're going to move this lever and it's going to close that loop to the column which means your sample then will get injected to the column so the sampling loop is an interchangeable loop it permits the introduction of samples at a pressure of 7000 psi so it allows for that so by using the loop versus using just like if you were just injected you do get increased precision the precision is generally a few tenths of a percent rsd so you get really high precision with using the loop you fill the loop the loop is full another common issue is overloading so if you just injected straight into the instrument you could have reproducibility issues and overloading but by using this loop it eliminates those as an issue because the loop is only holds a certain velocity and then your injection will be consistent every single time sample sizes usually are a few tenths of a microliter up to about 500 microliter and again that loop is interchangeable so you can have different size loops and it'll give you different volumes that get injected onto your column next we're talking about is going to be the column itself in hplc we work with two different types of columns so here's what a column looks like the first one is an analytical column this is one that's actually going to be used for detection of your sample so you can have different types of analytical columns that will allow you to detect four different things typically the length of these is going to be 10 to 30 centimeters the inner diameter 4 to 10 millimeters particle size inside is going to be 5 to 10 micrometers and the most common conditions used is 25 centimeters 4.6 millimeter inner diameter and a 5 micrometer particles this gives you about 40 000 to 60 000 plates per minute as i mentioned previously analytical columns are very expensive and they're reusable if you were to put something in let's say you have a contaminant in your sample and it gets to the column some stuff gets retained in the column and never goes off and as a result of this if something gets retained in the column and never officially comes off then every time you run a future sample you're going to see basically a contribution of that retained sample by an additional peak and so sometimes it's not an issue but sometimes that could be in the same spot as your sample and then you won't see your peak so to protect the analytical column we use a guard column they are much more cost effective you can replace them as you need to and this is always introduced before the analytical column to increase the life of the analytical column so this is going to remove particulate matter and contaminants from the solvent so if your solvent wasn't clean it's going to remove those before it goes to your analytical column it removes sample components that might bind irreversibly to the stationary phase and it serves to saturate the mobile phase with the stationary phase the composition of the guard column should be closely related to that of the analytical column but with larger particle sizes and this is to ensure that whatever your sample is is getting exposed to a similar composition and if something's going to be retained irreversibly then it gets retained in the guard column instead of the analytical column we have a couple different types of column packing that we can use the first one is a particular packing this is spherical non-porous glass or polymer beads with diameters of 30 to 40 micrometers you have a thin layer of silica alumina polystyrene um divinely benzene resin or an iron exchange resin deposited on the surface of a bead these are largely used for guard columns and not so much analytical columns the next type of packing is your porous particle packing here your porous particles have diameters of 3 to 10 micrometers and they're composed of silica alumina synthetic resin polystyrene divinely benzene or iron exchange resin silica is generally your most column you see in liquid chromatography so silica is your most common one that we would use there are two common types of detectors that we use the first one is a bulk property detector this one measures changes in a property that is typical of the solvent and solute as a whole some of those properties that it measures refractive index dielectric constant or density and this is modulated by the presence of solutes a solute property detector measures the concentration of compounds by measuring a property that is typical only to the compound solute in question and so those properties are going to be things like uv absorbance fluorescence or diffusion current this last slide is looking ultimately at different types of detectors you can use so it's different performances so if you want absorbance for your hplc detectors it's commercially available your limited protection is shown here in picograms this one's given in femtograms and your linear dynamic range so depending on which property you want to analyze at which type of limit detection you are looking for what's your linear dynamic range you can optimize or use different types of detectors or use what you have available to you