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 vs. Reversed Phase HPLC

Normal Reverse
Packing polarity High Low
Solvent polarity Low High
Non-polar first, then
Elution order Polar first, then non-polar
polar
Effect of increasing solvent Decreases retention
Increases retention time
polarity 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.
Hints for Good HPLC Injections
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.
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.