The Agilent 1200 HPLC is used for teaching and research and is located in room 109A. An HPLC system is unique among laboratory instruments because it can be assembled using components from different manufacturers and suppliers. Although many systems are sold as complete packages, a far greater number are assembled by bench level scientists and customized for specific needs. Pumps, detectors, columns, column ovens, and data management systems can all be interchanged. For example, when a pump is removed for service or the result of a breakdown, a new one can be swapped in with little or no interruption of service. Most components have their own keypad as well as a computer connection. The operator thus has a choice of running them off a central computer or via the keypads. Often the latter option proves the more convenient.
Compared with classical column chromatography, where the columns are gravity fed and a separation can take hours or even days, HPLC can offer analysis times of 5-30 minutes. Such times are comparable to that needed for GLC analyses.
HPLC is especially suited to the analysis of compounds not readily assayed by GLC. For example, thermally labile compounds can be analyzed by HPLC at ambient temperatures, and highly polar or involatile compounds can be analyzed. Sample treatment is often minimal since aqueous solutions can be used in HPLC.
Since its inception in the late 1960's, HPLC has made significant practical impact on the areas of pharmaceutical, clinical, forensic, environmental and industrial research and development analyses.
Columns
There are two main classes of column: "normal" and "reversed" phase. Normal phase columns are most usually packed with silica gel; they 
work in the partition/adsorption mode in the same manner as a normal silica gel column in conventional chromatography. Reversed phase chromatography, which is the most common form of HPLC, is a type of partition chromatography. Frequently, reversed phase columns are packed with a chemically bonded octadecylsilyl coated silica; such columns are referred to as C-18 and are very non-polar. Other popular bonded phase columns have octasilyl, cyanopropyl, or phenylsilyl packings. The eluent used with reversed phase columns is relatively polar, e.g. MeOH/H2O. Unlike normal phase chromatography, the more polar components of a mixture elute first, since these partition relatively unfavorably on the highly non-polar packing. Increasing the polarity of the solvent increases the retention time of a particular component. The situation is the reverse of normal adsorption chromatography:
| Normal | Reverse | |
|---|---|---|
| Packing polarity | High | Low |
| Solvent polarity | Low | High |
| Elution order | Non-polar first, then polar | Polar first, then non-polar |
| Effect of increasing solvent polarity | Decreases retention time | Increases retention time |
Step 1. Preparing the System Column – If the column mounted on the system is not the correct tthe requisite assay, pump an appropriate storage liquid through the column and removing it. Cap the ends and return the column to the correct drawer. A column rack helps keep columns organized and makes them easy to find.
Check the column's direction of flow. This is identified by an arrow stamped on the side of the column or marked on the label. After installing the correct column, tighten the connections so they do not leak. Firm but gentle pressure should be applied to the connections. Excessive force is not required.
Mobile Phase – The mobile phase must be free of dissolved gasses so that no bubbles form inside the instrument during the run. Each system in the chromatography laboratory has a connection to a helium tank. Gently bubbling helium through the mobile phase for a few minutes will generally remove all dissolved gasses. Some laboratories use sonic baths to degas the mobile phases and many new HPLC systems are sold with degassing units built into the pump. Often the mobile phase is mixed in large quantities. When preparing a mobile phase with two or more components, be aware that large amounts of heat can be generated by the mixing process. Glass bottles may crack! The selection of mobile phases is based on the relative polarity of the analytes and the column packing. Often the addition of just a few milliliters of some ionic species will dramatically affect the analysis. For example, 20 mM triethylamine in the mobile phase will reduce certain types of peak tailing. Adding acetic acid to a mobile phase will increase its overall polarity and result in better separations when running on a non polar stationary phase. Controlling the mobile phase's pH by the addition of acid, buffer, or base will often improve reproducibility.
Step 2. Prime the pump. Select a mobile phase by either placing the intake line in the bottle or using the keypad on the pump to select a specific bottle. Open the “prime” or the “purge” valve on the pump module. Place a beaker under the outlet . Activate the “prime” or “purge” function on the pump. If the pump does not have this function, turn up the flow and switch it on.Run the system for a few minutes. When the pump is properly primed, the system will deliver a smooth flow of mobile phase from the outlet, produce a steady sound with no burping or grinding, and the inlet line will be free of air bubbles.Turn off the pump and close the valve.
Step 3. Set the detector wavelength. Turn on the detector power and allow the unit to warm up. Using the keypad or the control computer GUI, set the wavelength(s).
Step 4. Start the mobile phase flow. Use the pump controller, to set the flow rate for the mobile phase . Restart the pump. Watch the system's pressure indicator or gauges to see that it does not exceed the maximum allowed for the various components. If pressures become too high, slow down the flow rate.
If pressure continues to rise, turn off the pump, and perform the following procedures:
a. If the system has a guard column, replace it.
b. The analytical column may need to be back flushed. Remove the column and reverse it so that mobile phase flows through it in the wrong direction. Catch the outflow in a beaker instead of allowing it to enter the detector.To condition the column, run mobile phase through it for a few minutes. Some operators recycle their mobile phase by running the waste line outlet back into their mobile phase reservoir. Care must be taken to avoid contaminating the reservoir with old samples, or impurities from the column. Monitor the detector output, when the signal is stable, begin running the samples.
Step 5. Inject the samples. Before injecting a sample, check the needle's tip. HPLC Needles have a smooth or blunt tip. Do not use a needle with a sharp tip or a tip with metal burrs. These will scratch the inner surfaces of the injector and cause it to leak. Remember this is a two-step process; syringe injection followed by turning the valve from "load" to "inject." Open the injection valves by turning them to “load.” Insert needle into the plastic needle guide as far as it will go. Smoothly inject the appropriate amount of sample. After the syringe is completely empty, quickly and smoothly turn the valve to “inject.” It is safe to leave the syringe in place. Start the data system recording. Behind each injector, there is a small coil of tubing. Known as the "sample loop," it holds the sample during the interval between the syringe injection and the start of the run. The sample loop can be identified by a small tag listing its volume. When the injector is set to the 'load" position this loop is isolated from the mobile phase flow. Turning the valve to "inject" diverts the mobile phase flow through the sample loop and sweeps the sample onto the column. For manual injections, as long as the sample volume is less than or equal to the loop volume, changing the loop is not necessary. More experienced with HPLC will teach how to determine a good injection volume. Column capacity, detector sensitivity, and column size, must all be balanced to obtain the best results.
Make sure there are no air bubbles in the syringe. Even a small bubble can affect the injection volume. Also make sure that any excess sample is wiped off the exterior of the needle.
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|---|
| Insert the needle completely into the injector's plastic sleeve. Do not press the plunger until the needle can go no further. |
| Inject completely before turning the valve, but do not wait more than a few seconds between these two operations. |
Step 6. After the run. Stop the data station . Return the injector to the "load" position and remove the syringe.
Step 7. Cleanup. Turn off the detector, pump, and any other components. Rinse any sample residues from the syringe. Record the sample date and initials in the appropriate notebook. Label the chromatogram with:
1. The users name, date, and sample ID.
2. The column, mobile phase, flow rate, and detector wavelength.
3. The system number and its calibration date.
Step 8. Is this data any good? Evaluate the chromatogram, look for:
1. Good separation. Is the first analyte of interest sufficiently separated from the solvent front? Are all analytes separated or are peaks running into each other?
2. Peak broadening and tailing. Broad peaks are difficult to quantify. Excessive tailing can hide minor peaks.
3. Irregular retention times. Retention times should not shift from one run to the next although small shifts caused by operator variability are normal.
4. Flat and level baselines. Undulating, irregular, or jagged baselines indicate contamination of the column, carry over between injections, or excessive sample loading. Similar symptoms can result from problems with the detector.
Operation of the HPLC - General Procedure
Cartridge: 8 x 100 mm m-Bondapak C18 (a reversed phase column).
Solvent: 0.5% phosphoric acid in 40% aqueous methanol.
The greatest enemy of HPLC is fine particulate matter, which can damage the pumping system and irreversibly block the column. Therefore, all solvents have been filtered through fine membranes (0.45 micron) and all solutions to be injected MUST be prepared either with filtered solvent, or filtered as specified later in these notes.
The instrument comprises three main components: the injector, the solvent delivery system and the detector.
Start-up Procedure
First, ensure that there is sufficient filtered solvent in the reservoir for a run.
Using the screw pressurize the column to 800 psi. Switch the solvent selector on the inlet manifold at the front of the pump to the running solvent. Switch on the power to the pump and slowly increase the flow rate to 3 mL/min. Switch on the detector and once the absorbance reading has settled (~10 min), set the zero to 0.01 AU. Switch on the Data Module and enter the date and time (e.g. 2007/07/28/ 09/2/) and answer 0/ to "Is this a new file?"
To inject a sample: Switch the injector lever (top) to "load". Switch the lower lever to vertical and remove the plug (store in hole in injector switch). Wipe the needle with a clean tissue and insert into the injector. Inject the sample into the loop with even pressure (excess of solvent in the loop will be pushed out of the vent tube on the right of the injector). Replace the injector plug and move the low lever to the horizontal position. Smoothly switch the injector lever to "inject", and at the same time press "inject" on the data module to start the data collection. The data module plots a real time chromatogram, and at the end of the run time (15 min) replots the chromatogram with details of retention time (RT), peak area (A or H) and relative areas of the peaks (conc). Although the integration is not affected if a plotted peak is off scale, the chromatogram can be replotted at a different attenuation by resetting the ATN (powers of 2, the bigger the attenuation setting the smaller the peaks will look, normally set at 30) and then recalling the plot (Recal). The plot is stored until the next injection.
Shut down procedure
Stop the flow slowly and change the solvent selector to flushing solvent (methanol). Increase the flow to 3mL/min slowly and flush the column with solvent for 10 min. Stop the flow and switch off the pump. Remove the plots from the Data module and switch off at the front of the unit. Switch off the detector. Depressurize the column by unscrewing the pressure screw on the right of the RCM until 4 threads are showing.
Notes: Normal running pressure for this experiment is between 1500-2200 psi. If a high pressure shutdown occurs (>2500 psi). Look for leaks at connections through the system.
Listen to the pump during the experiment; if there are any unusual noises. Look at the outlet flow; it should be a thin stream.
DIODE ARRAY
Agilent Technologies has a new 1200 Series diode-array and multiple-wavelength detectors featuring improved noise specifications. These liquid chromatography detectors allow lower detection limits, even under harsh, fluctuating ambient temperature and humidity conditions.
The Agilent 1200 Series diode-array and multiple-wavelength detectors additionally offer:
* Dual lamp design for highest sensitivity from 190 to 950 nm;
* Programmable slit for easy optimization of sensitivity, linearity and spectral resolution;
* Low-noise electronics and electronic temperature control for lowest detection limits, even under unstable ambient conditions
* A range of nine flow cells for highest application flexibility; and
* Easy upgrade to the 80 Hz sampling rate for high speed separations.
* The new detectors reinforce the scalable and open architecture of the 1200 Series LC platform.
The new detectors reinforce the scalable and open architecture of the 1200 Series LC platform,