HPLC Procedure for Testing Sugars
Philyaw; Senior Lab Project Spring 2004
1. First step is to prime the reference cell.
This can be done by locating the outlet for the mobile phase. The outlet is
noted by the metal screw type cap found on one of the lines connected to the
refractive index unit. The reference cell lines are both capped with plastic
ends. Remove the metal screw cap from the outlet line. This can be done
without tools. Now remove both of the plastic caps from the reference lines
and replace one of them with the metal screw cap. In the top of the metal
screw cap is a plastic plug. Remove this plug by unscrewing it and set it
aside. A special syringe will screw into the top of this metal cap where you
removed the plastic plug. Locate and fill the syringe with a small amount of
the mobile phase, then screw it into the metal cap. Flush until the fluid comes
out of the reference cell outlet. Most mobile phases are toxic so be sure to
have some kind of vessel to catch the excess fluid. Once this is done, cap the
outlet end with the plastic cap removed earlier and replace the plastic plug
removed from the metal screw cap to prevent evaporation. If many samples
need to be analyzed, the reference cell may need to be primed again after
several hours of running.
2. Next, verify that all units have power
Make sure the power strip is on. Turn on the refractive index unit by pushing
the power button in. A light should illuminate the button noting the unit is on.
Next, plug in the column. There is no on or off button so, once the column is
plugged in, it is on. The column is attached to a heater. To set the desired
temperature of the column, lift up the box unit the column sits in and flip it
over so you can see the back. *Note: the plastic cover for the column is
magnetized and may fall off when flipping the unit over. On the back of the
unit, there are arrows that allow the user to change the temperature up and
down as necessary. Once the temperature is set, then place the unit back in its
holder. Now, set the pump flow to 0.5 ml/min and flip the switch to on.
*Note: be sure that a vessel in place to catch waste mobile phase before
turning the pump on! Fluid should start coming out of the outlet line with in a
minute of starting the pump. If fluid does not come out after a minute, there
may be a clog or a leak. Refer to the manufacture’s information if the filter
needs to be replaced or leaks occur.
3. Flushing the system
Now that all the units are running, let the mobile phase flow through the
system for a minimum of 30 minutes before running the first sample. This
will ensure that anything left in the column from previous experiments is
cleaned out, in addition to giving the refractive index unit time to properly
warm up.
4. Setting up the computer program
Log on to the computer sitting next to the HPLC unit with the username and
password specified on the computer tower. On the desktop is an icon called
DIAMIR. Double click on the icon to open the program. A prompt will ask
you for a password, type in the same password to log on to the computer.
Once the program is open, a method must be created. Follow the DIAMIR
file instructions located next to the computer. The method allows the user to
specify sample run time in addition to sample information.
Note if the
retention time of the samples is not known, it is better to have a longer run
time to ensure elution of the sample. The user can manually stop the program
at anytime, but cannot extend the time frame once the program is running
without going back into the method.
5. Sample preparation
Samples are first filtered with 0.2-micrometer syringe filters to ensure all
bacteria, yeast, or any other foreign particles are captured. Note that these
particles can clog and/or damage the column and must be removed before
injecting the sample. Rinse the injecting syringe at least three times with the
sample to ensure no residual remains from the previous sample. Draw a
slightly larger amount of the sample into the injecting syringe and gently push
out the excess. Dab the tip of the syringe on a Kimwipe® to dry the excess
and make sure no bubbles exist in the syringe.
6. Preparing the software for acquisition, baseline monitoring and sample
runs.
Before running samples and after the equipment has properly warmed up, the
baseline must be checked to ensure proper detection of samples. At the top of
the DIAMIR, program select acquisitionmonitoring baseline.
A
chromatograph should appear. If it does not appear, look on the lower left
corner there should be three tabs. Choose the system tab. At the top left
should now be a title that says Waters or HPLC. Click on the plus sign next to
this title, and then click in the boxes below that say status and chromatograph
so that they now have a check beside each. Once the chromatograph is visible,
change the range so that the baseline is visible by choosing the far left icon.
This will open a menu containing two tabs. Choose the bottom tab, uncheck
the boxes next to the range, and close. Now the baseline should be visible on
a larger scale. If the baseline is not trending on zero, then turn the optical zero
knob on the refractive index unit while you watch the baseline until it is stable
and close to zero.
Clockwise turns of the optical zero knob makes the
baseline trend down and counterclockwise turns trends the baseline up. It
does not have to be perfectly on zero but the closer the better. Most important
is that the baseline is steady and not drifting up or down.
Now to set up to run samples: At the top of the DIAMIR, program select
acquisition quick start. A drop down box will appear, choose the method
created earlier. Next a sample identification menu will appear. The first line
will note the name of the file, so make it specific to the sample. Other things
to note are flow rate, sample concentration, mobile phase concentration, etc.
Once this is complete, hit enter. This will bring up a new page that should say
waiting for injection. If this page does not appear, look on the lower left
corner there should be three tabs. Choose the system tab. At the top left
should now be a title that says Waters or HPLC. Click on the plus sign next to
this title then click in the boxes below that say status and chromatograph.
Now the page that says waiting for injection should be visible. The program
will automatically start once the sample has been injected.
7. Injecting the sample
Now that the DIAMIR program is waiting for injection, make sure the switch
on the injection port is on load and not inject. Now draw the sample into the
injection syringe as described in step 5, and then release the injection port by
turning the lever down. Pull out the round silver plug and place it on a clean
Kimwipe® on top of the pump unit. These next steps should be done quickly.
This will only come with practice so don’t be alarmed. Inject the sample into
the port, place the round plug back into the port and lock it by turning the
lever up until it is tight. IF YOU DO NOT TIGHTEN THE PLUG, IT CAN
SHOOT OUT WITH THE FORCE EQUAL TO THE BACK PRESSURE!
THIS IS VERY DANGEREOUS SO DON”T FORGET THIS STEP! Next,
turn the switch from load to inject. You will notice that DIAMIR will start as
soon as you switch it to inject. Wait until you see 0.3 minutes on the DIMAIR
timer and turn the switch back to load. The user may manually stop the
program when all the components have eluted (usually by 12 minutes) by
clicking on the red icon located at the top of the chromatograph window
containing an X.
8. Printing your Chromatograph
In the DIAMIR program window choose file open Chromatograph. A
window will appear with a list of files; choose the particular file by double
clicking on it. It should open revealing the chromatograph. Now choose the
results option just below the method string located at the top left menu box.
Retention times, area percent, and start/stop times should be listed below the
chromatograph.
Injection Port
Pump
Luna
Column
Refractive
Index Unit
Figure 5 - HPLC apparatus
DIAMIR
Program