EXPERIMENT 18
LIQUID CHROMATOGRAPHY
Chromatography is a technique by which a mixture sample is separated into
components. This technique requires that a solute undergo to distribution between two
phases, one is fixed (Stationary phase), the other is moving (mobile phase). It is used to
separate and recover (isolate and purify). The components of a sample, and also , are
often used to both separate and quantify. The types of chromatography used in
qualitative and quantitative analysis are column, gas, paper, thin-layer, and high
pressure liquid chromatography or high performance liquid chromatography.
The HPLC is separation based on a solid stationary phase and liquid mobile phase. The
equipment consists of a reservoir containing a mobile phase. A pump to force the
mobile phase through the system at high pressure , and injector to introduce the sample
into the mobile phase , a chromatographic column , a detector and a data collection
device , as shown below.
OBJECTIVE:To be able to perform quantitative analysis using High pressure Liquid
Chromatography.
APPARATUS / GLASSWARE: HPLC (Model LC-10AS, Shimadzu) or any available
brand, reagent bottle for mobile phase, HPLC-grade reagents
MATERIALS / SAMPLES: (To be Assigned by the Instructor)
PROCEDURE:
1. Turn on AVR/UPS
2. Turn on the PUMP ( LC-10AS/LC-10AT)
2.1. Immerse solvent , filter in appropriate mobile phase
2.2. Open drain valve
2.3. Purge the pump (Press PURGE button for auto cycle or do manual
purging with syringe.
2.4. Close drain valve.
2.5. Set appropriate flow rate.
2.6. Condition the system until stable pressure is obtained ( Kg Cm² reading
should not fluctuate by more than 4 units.
2.6.1. IMPORTANT!
2.6.2. If mobile phase to be used is buffered or has some salt components,
perform step 1-6 using filtered, degassed, de-ionized/ distilled water
BEFORE and AFTER using the analytical mobile phase ! Run system
until the analytical total water flow equals about 5 times the column
volume
2.6.3. For C18 (Also LI or reversed-phase columns), always store the column
in methanol or methanol: water , (50 : 50)
2.6.4. After washing with water, run methanol through the column until all
water has been displaced. NEVER leave the column filled with water
only!
2.6.5. During washing, all other modules need not be turned on.
6.5. MONITOR BASELINE
6.5.1. Click TEST ZERO≫ ZERO
6.5.2. Monitor baseline for 5-10 minutes
6.5.3. if stable, Click TEST/ZERO≫SLOPE TEST
6.5.4. If slope value from 0-300, click OK.
6.5.5. ( continue to monitor baseline if, value is too high)
7. SPECIFY STD/SPL DATA INFORMATION
7.1. Click sample login and fill in the necessary information.
7.2. If set filename already exists, change name or extension name if you do not
wish overwrite the old date.
7.3. Click Login
7.4. Inject standard solution. Data collection should start automatically once injector
is set to inject position.
8. INJECTION PROCEDURE
[Link] injector to inject position
8.2. Insert syringe
8.3. Set injector to load position
8.4. Press syringe
8.5. Set injector to inject position
8.6. Wash injection port
9. POST-RUN ANALYSIS
9.1 Minimize Real time analysis window
[Link]. Select POST RUN
[Link]. Select SINGLE CHROM
[Link]. Select FILE
[Link]. Load data files ( or data file of standard analyzed at start)
9.2. Select EDIT, QUANTITATION, and change the following:
9.2.1. Method – External
9.2.2. Minimum Area – As desired
9.2.3. Window% As desired (e.g 2%)
9.2.4. Click OK
9.2.5. Fill-in the Quantitation Table
9.2.6. Input the retention time (type in the value or drag the blue arrow to
the top of the analyte peak)
9.2.7. Type in the components name, concentration.
9.2.8. Click OK
9.3. Select EDIT, TIME PROGRAM (input necessary)
9.3.1. Changes as desired, e.g. to eliminate extraneous peak)
[Link]. Select PROCESS
9.3.2. Select CALIBRATE or INTEGRATE
9.3.3. Click Yes/OK
9.4. Select FILE, click SAVE DATA
9.4.1. Select FILE, Click SAVE METHOD
9.4.2. Click Yes and close POST RUN ANALYSIS window
10. INJECT STANDARDS AND SAMPLES
10.1. Maximize REAL TIME ANALYSIS WINDOW
10.1.1. Click SAMPLE LOGIN
10.1.2. Register standard name and inject standard solution
10.2. Type in desired no. of repetitions
10.3. Calibration curve is printed after all report for standards.
10.4. Click SAMPLE LOGIN
10.5. Register sample name and inject sample solution
10.6. Type in desired no. of repetitions.
11. SHUT-DOWN
11.1. Close the GC-10 Window
11.2. Click start. Select shutdown
11.3. Turn –off PC
11.4. Turn- off CBM
11.5. Turn – off Detector
11.6. Wash HPLC system with appropriate wash solvent (e.g. water if column is –
reversed-phase and buffered mobile phase was used about 30 mins. Then methanol
or methanol:water.
11.7. After washing, turn off pump, then UPS/AVR.
