HPLC Procedure for Testing SugarsPhilyaw; Senior Lab Project Spring 20041. First step is to prime the reference cell. This can be done by locating the outlet for the mobile phase. The outlet isnoted by the metal screw type cap found on one of the lines connected to therefractive index unit. The reference cell lines are both capped with plasticends. Remove the metal screw cap from the outlet line. This can be donewithout tools. Now remove both of the plastic caps from the reference linesand replace one of them with the metal screw cap. In the top of the metalscrew cap is a plastic plug. Remove this plug by unscrewing it and set itaside. A special syringe will screw into the top of this metal cap where youremoved the plastic plug. Locate and fill the syringe with a small amount ofthe mobile phase, then screw it into the metal cap. Flush until the fluid comesout of the reference cell outlet. Most mobile phases are toxic so be sure tohave some kind of vessel to catch the excess fluid. Once this is done, cap theoutlet end with the plastic cap removed earlier and replace the plastic plugremoved from the metal screw cap to prevent evaporation. If many samplesneed to be analyzed, the reference cell may need to be primed again afterseveral hours of running. 2. Next, verify that all units have powerMake sure the power strip is on. Turn on the refractive index unit by pushingthe 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 isplugged in, it is on. The column is attached to a heater. To set the desiredtemperature of the column, lift up the box unit the column sits in and flip itover so you can see the back. *Note: the plastic cover for the column ismagnetized and may fall off when flipping the unit over. On the back of theunit, there are arrows that allow the user to change the temperature up anddown as necessary. Once the temperature is set, then place the unit back in itsholder. 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 beforeturning the pump on! Fluid should start coming out of the outlet line with in aminute of starting the pump. If fluid does not come out after a minute, theremay be a clog or a leak. Refer to the manufacture’s information if the filterneeds to be replaced or leaks occur.3. Flushing the systemNow that all the units are running, let the mobile phase flow through thesystem for a minimum of 30 minutes before running the first sample. Thiswill ensure that anything left in the column from previous experiments iscleaned out, in addition to giving the refractive index unit time to properlywarm up. 4. Setting up the computer programLog on to the computer sitting next to the HPLC unit with the username andpassword specified on the computer tower. On the desktop is an icon calledDIAMIR. Double click on the icon to open the program. A prompt will askyou 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 DIAMIRfile instructions located next to the computer. The method allows the user tospecify sample run time in addition to sample information. Note if theretention time of the samples is not known, it is better to have a longer runtime to ensure elution of the sample. The user can manually stop the programat anytime, but cannot extend the time frame once the program is runningwithout going back into the method.5. Sample preparationSamples are first filtered with 0.2-micrometer syringe filters to ensure allbacteria, yeast, or any other foreign particles are captured. Note that theseparticles can clog and/or damage the column and must be removed beforeinjecting the sample. Rinse the injecting syringe at least three times with thesample to ensure no residual remains from the previous sample. Draw aslightly larger amount of the sample into the injecting syringe and gently pushout the excess. Dab the tip of the syringe on a Kimwipe® to dry the excessand make sure no bubbles exist in the syringe.6. Preparing the software for acquisition, baseline monitoring and sampleruns.Before running samples and after the equipment has properly warmed up, thebaseline must be checked to ensure proper detection of samples. At the top ofthe DIAMIR, program select acquisitionmonitoring baseline. Achromatograph should appear. If it does not appear, look on the lower leftcorner there should be three tabs. Choose the system tab. At the top leftshould now be a title that says Waters or HPLC. Click on the plus sign next tothis title, and then click in the boxes below that say status and chromatographso 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, uncheckthe boxes next to the range, and close. Now the baseline should be visible ona larger scale. If the baseline is not trending on zero, then turn the optical zeroknob on the refractive index unit while you watch the baseline until it is stableand close to zero. Clockwise turns of the optical zero knob makes thebaseline trend down and counterclockwise turns trends the baseline up. Itdoes not have to be perfectly on zero but the closer the better. Most importantis 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 selectacquisition quick start. A drop down box will appear, choose the methodcreated earlier. Next a sample identification menu will appear. The first linewill note the name of the file, so make it specific to the sample. Other thingsto 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 saywaiting for injection. If this page does not appear, look on the lower leftcorner there should be three tabs. Choose the system tab. At the top leftshould now be a title that says Waters or HPLC. Click on the plus sign next tothis title then click in the boxes below that say status and chromatograph.Now the page that says waiting for injection should be visible. The programwill