Used HPLC System in Chromatography
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Waters ACQUITY UPLC System
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Modular High Pressure Liquid Chromatography (HPLC) systems are generally used for the separation of analytes in aqueous mediums. Because of the modularity of Agilent HPLC’s, they are easy to work on and can be configured in many different ways.
The components on an HPLC can consist of the combinations of pieces below, and are often referred to together as the HPLC stack. What is in the stack can vary, but at its most basic the components consists of a pump, injector, column and detector.
Anatomy of an HPLC Stack
Below is a short description of the Agilent HPLC components with the corresponding “G” number that Agilent uses as the model number for that item., for instance G1322A is a vacuum degasser, whereas G1379B is a micro vacuum degasser. The HPLC stack can consist of any of the following items.
The first item is generally a solvent tray to hold the solvent bottles. While this item is not necessary, it is nice to have and keep your solvents organized in one place.
The second item is a vacuum degasser. These come in both regular and micro degasser configurations (G1322A, G1379B). Degassers remove dissolved gas trapped in liquids prior to the solvent being feed into the high pressure pumps. This reduces the risk of bubble formation later in the HPLC flow.
Preparing and Controlling the Mobile Phase – Solvent Pumps
Next is the HPLC pump. The pump is one of the “must have” components of the system, and generally runs at pressures of 2500 to 4500 psi or more. Pumps come in many different configurations depending on what is required. The first determination you must make is if you are doing the separation isocratically (G1310A), i.e. using the same solvent throughout the entire run, or using a gradient which changes the solvent characteristics over time. A gradient HPLC is the most common method of running a HPLC, and the gradient is accomplished using either a binary pump (G1312A) with two solvents max, or quaternary pump (G1311A) with four solvents max. Technically you can use a binary or quaternary pump to run an isocratic solvent by maintaining a constant ratio of the selected solvents throughout the run; this is one reason why an isocratic pump is the least favored of the three, it is not as versatile as the others.
Another consideration is the purpose of the separation in regards to flow rates of the pump. Standard analytical columns run at 1.0 ml/min. If you have very small amounts of sample you can increase the efficiency of the separation by using either capillary or nano HPLC. As the name suggests capillary pumps (G1382A) run at a reduced flow rate of 1-100ul/min, and nano pumps (G2225A) run at even lower rates of 0.1-4ul/min. Nano pumps are used primarily for mass specs.
On the other end of the spectrum are preparative pumps (G1361A), with pump rates of 5-100 ml/min, and with some manufacturers significantly higher. A prep pump is generally used when a compound requires isolation in larger quantities. The separation is collected in a fraction collector at the end instead of diverted to waste.
Introducing the Sample – Injectors
The next item in the stack is an injector, the piece that allows the analytes or solution to be inserted into the HPLC flow path for separation on the column and eventual detection by the detector. The simplest version of this is a manual injector, such as a Rheodyne or other brand. The next level up is the automated sample injector that allows unattended injection of many different samples. Using an auto-injector tends to increase run reproducibility compared to a manual injector. Autoinjectors alo come in different models, such as the analytical (G1329A) or the 96 well plate sampler (G1367A). The amount of the injection is controlled in one of two ways, depending on if you are using a manual or automatic injector. In a manual system you select the sample length from a variety of injection loops available by the manufacturer. For instance, with a Rheodyne manual injector you might choose a 5ul loop, 10 ul loop, etc. This allows for the same repeated injection volume. Changing the injection voume requires manually switching the sample loop for a different size. With an automatic injector the system uses a syringe driven by the computer or injector programming panel. Changing sample size is a matter of typing in the new injection volume, and the syringe draws the correct amount. As an example, a standard syringe may be 250ul in volume, allowing the autosampler to inject volumes between 1-250ul, simply by filling the syringe with the correct amount of sample. This is extremely convenient formethod development and other instances where sample injection size might vary.
A nice component to have with an automatic sample injector is a temperature controller. Agilent makes the thermostat control unit (G1330B) that allows either heating or cooling of you samples. Keeping samples at 4 degrees C or some other temperature can prevent degradation of sample over time. When HPLC autoinjectors are loaded up with large amount of samples for automatic overnight runs this temperature control may be critical for sample integrity.
Where the Separation Occurs – Columns
Once the sample is injected it goes directly into the HPLC column, although a precolumn can be used just in front of the column. Precolumns can be either the same or of a different composition then the actual column used for the separation. Generally precolumns are considered cheap protection devices for the actual columns, sort of a filter to prevent the main column from clogging up early.
The column is a disposable piece that accomplishes the separation. Columns vary significantly in both size and composition, with many different brands available, allowing for an almost infinite variety of separation possibilities. Even columns that are labeled in identical manner from one manufacturer to another can give very different results, in part due to trade secret formulations and processing technologies.
The columns used to separate mixtures of components are usually made up of a silica based microspheres with carbon chains on them, packaged inside a steel column strong enough to withstand the pressures of an HPLC system. The carbon chain attachments can vary, but the most popular are referred to as C18 columns, having 18 CH3 molecules attached to the silica microsphere. The columns can come from a wide variety of manufacturers. Other chemistries besides carbon based are also available.
Another nice item to have for the HPLC stack is a column temperature control unit (G1316A). This device houses the column (and precolumn) in a temperature controlled environment, usually between 30-40 degrees C. This tends to accomplish a number of things: A) increases the repeatability between runs since the column is not subject to room temperature fluctuations, B) shortens the run time slightly, and C) most importantly it can help sharpen your peaks creating a better chromatogram and cleaner isolaton.
Finding the Analytes – Detectors
The final must have item in the HPLC stack is the detector. The detector is immediately after the HPLC column and has a continuous stream of fluid passing through the flow cell. The flow cell is where the detection occurs. A beam of some type is passed at right angles through the flow cell with a beam detector on the other side of the flow cell. As compounds pass through the flow cell from the HPLC stream, that beam is deflected or absorbed, and that difference in the beam strength is measured on te detector side. This difference in beam strength is what is shown on the chromatogram. Detectors come in the largest variety of configurations, and selection of the correct detector is key to a successful separation. Detector selection is based on what characteristics the compound that is being analyzed has. One of the most basic detectors is based on UV light absorption, so if your analyte absorbs UV light you can use a UV detector in your HPLC. Example: Peptide separations are generally preformed usin a UV detector set for 214nm, because peptides absorb well in this spectrum.
Agilent makes several detectors in this range. The Variable Wavelength detector (G1314A) allows an operator to pick the wavelengths they need. A multi-wavelength detector (G1365A) allows the operator to pick multiple wavelengths for acquisition at the same time. A Diode array detector (G1315A) has a diode array as the acquisition source, allowing a spectrum of wavelengths to be acquired all at the same time. This detector allows for 3-D plots of the analyte flowing though the flow cell, giving a finer print quality to the analyte. Another detector type is the fluorescent detector (G1321A) which picks up only molecules that fluoresce. With the more advanced detectors in this area the specific excitation and emission wavelengths of the fluorescent item are selected in the detector by the operator further helping the detection of the analyte.
There are also detectors that are not based on any wavelength, such as radioactive detectors, although Agilent does not make one of these. Electrochemical detectors are made that can measure the electrical chemistry of molecules as they flow by the flow cell. RI or refractive index detectors (G1362A) are used for compounds that have none of the above detection abilities. Infrared detectors are available for molecules that have IR qualities.
Recovering the Analytes – Fraction Collectors
The last item you might have in an HPLC stack is the fraction collector (G1364C). This is allows the eluent coming from the detector to be collected in test tubes or microfuge tubes rather than going into a waste container. The eluent stream from the HPLC can alternatively be directed into different test tubes depending on either set time interval, or the slope of a peak on the chromatogram. Slope detection allows the fraction collector to switch test tubes from one peak to the next, giving the purestisolation of the analyte compared to a set time system. Both have their pros and cons, as slope detection is a computer algorithm and may or may not catch your peak at the right moment. For highly important samples it is advisable for the operator to watch the progress of the HPLC separation and manually control the fraction collector. Once the samples of interest are collected the fraction collector is diverted to a waste bottle for the rest of the run.
These are the basic components of an HPLC stack. A stack can vary substantially in composition and therefore in price. Since HPLCs vary significantly depending on what they are designed to do, the end result is difficult to compare when shopping from one HPLC to another. As an example, a VWD detector is about half the price of a DAD detector. Does the HPLC stack you have consist of the basics of pump, injector and detector, or is it a fully outfitted stack with degasser, injector temperature contro unit etc.?
Please see below for examples of the Agilent HPLC systems for sale at BioSurplus. Please contact a sales representative for more information about purchasing one of them, at 866-236-4496, x201.