• Embed Doc
  • Copy Link
  • Readcast
  • Collections
  • 2
    CommentGo Back
Download
 
High Performance Liquid Chromatography - Chem 421
Prof. S. Shippy and K. Thongkhao-On, 3/24/07
Determination of Amino acids in Food by High Pressure Liquid Chromatography
High performance liquid chromatography (HPLC) makes use of a pump to deliver amobile phase solvent at a uniform rate at pressures that are typically from 500 to 5000 psi. Themost obvious advantage of HPLC over gravity liquid chromatography is that samples that are notvolatile or that would thermally decompose in gas chromatography can be rapidly and routinelyseparated. Consequently, this powerful analytical method is complementary to a gaschromatography. HPLC is accomplished by injection of a small amount of liquid sample into amoving stream of liquid (termed the mobile phase) that passes through a column packed with particles of a stationary phase. As in gas chromatography, separation of a mixture into itscomponents depends on different degrees of retention of each component in the column. Theextent to which a component is retained in the column is determined by its partitioning betweenthe liquid mobile phase and the stationary phase. A variety of HPLC separation techniques thatutilize different stationary and mobile phases have been developed.
Adsorption Chromatography
Adsorption chromatography utilizes a solid stationary phase of a polar nature such as particles of hydrated silica or alumina. The mobile phase and the solute (components of the sample) are incompetition for active adsorption sites on the stationary phase particles. Thus, more stronglyabsorbed components are retained longer than weakly absorbed components. Because more polar compounds absorb on a polar surface to a greater degree than do less polar compounds, retentionin the column is related to sample polarity. A Generalized polarity scale for various classes of compounds is shown in Table 1.
Partition Chromatography
This form of HPLC partitions the solute between the liquid mobile phase and a secondimmiscible liquid that is coated on or bonded to a solid particles as the stationary phase.Compounds that partition more strongly into the stationary liquid phase are retained longer in thecolumn. This type of chromatography is termed
normal phase
if the stationary phase is more polar than the mobile phase and
reverse phase
if the mobile phase is more polar than thestationary phase. The stationary phase can be a liquid coated on a solid support particles.
 Bonded  phase
columns have a stationary phase chemically bonded to the solid support and are the most popular column for partition chromatography. Fro example, n-octadecane can be bonded directlyto silica by attachment to surface hydroxyl group to form what is termed a C
18
column. Theextent of partitioning of a solute into the stationary phase can be controlled by varying thesolvent polarity.
2
 
Ion-Exchange Chromatography
This type of HPLC is based on the partition of ions between a polar liquid phase and a stationary phase with ion-exchange sites. The ion-exchange sires are typically immobilized in a small beadsof resin that are formed by a cross-linked polymer. Bonded phase columns in which the ionexchanger is bonded to small particles of silica are also available. Cations are separated on cation
 
exchanger resins which contain negatively charged functional groups such as -SO
3
- and -COO-.Anions are separated on anion exchange resin which contain positively charged functionalgroups such as -CH
2
 N(CH
3
)
3
, a quaternary ammonium ion. Separation is based on ions partitioning into the ion-exchange phase to varying degrees. The selectively of a resin for an ionis determined primarily by the charge on the ion and its hydrated radius. Resin affinity increaseswith increasing charge density.
Apparatus
In this experiment, a HPLC instrument that consists of a Waters 600E System with a WatersAbsorbance Detector is used for analysis. Figure 13.2 shows a typical HPLC system. The‘Waters 600 Controller’ controls the eluent gradient, flow rate, external events, and sparking for the Waters 600E System. It also provides connection terminals and communication ports for operation with and control of external devices, such as detectors, autosamplers, and data systems.The ‘Waters 600E Pump contains the components required to blend and to deliver eluents formthe eluent reservoir bottles to the injector and column. It consists of the pump with 225 ìL or 100ìL volume pump heads, eluent sparge valve assembly, eluent proportioning valve assembly,manual injector, and vent valve. The manual injector connects and disconnects the sampleloading loop from the system. It allows variable-volume injections from a fraction of a ìL to a 20ìL without changing the loop. The Waters 2487 Dual ë Absorbance Detector is a two channel,tunable, ultraviolet/.visible (UV/Vis) detector designed for high-performance liquidchromatography (HPLC) applications. Figure 14.19 shows a diagram of an absorbance detector for use with HPLC.
Reagents
In this experiment, amino acid standards will be prepared from a dilution of the 10 mM standardin mobile phase. The student will be provided a vitamin pill and the student will be required to bring in fresh fruit juice or honey.
Qualitative and Quantitative Measurements
A typical liquid chromatogram is shown in Figure 13.3 and 13.6. Each component in a mixturecan be qualitatively identified by its retention time t
R 
, which is the time between injection anddetection. As with gas chromatography, the retention time of a particular compound is constantfor a fixed set of chromatographic conditions (flow rate, temperature, column condition).Qualitiative identification is made by comparing t
R 
of the unknown with retention times of standards that have been injected into the chromatograph. This strategy works so long ascomponents have unique retention times.
3
The area under each peak is proportional to the concentration of that component in the originalmixture. If the peaks are reasonably sharp and the flow rate is carefully controlled, the peak heights are approximately proportional to concentration. Thus, a calibration curve can be prepared by plotting either height or peak area as a function of concentration for a series of standards. Methods for the measurements of peak areas are described in Figure 13.7.
Operation of Waters HPLC via Empower ProgramStart Up Procedure
 
1. Turn on power to PC hard drive2. Turn on power to PC monitor, log in and password are”chem421”3. Turn on power to “Waters 2487 Dual ë Absorbance Detector” wait ~6 min to warm up andsystem checking4. Turn on power to “Waters 600 Controller”–> Please wait for few minutes for warm up5. Double click ‘Start Empower’ icon that is on the desktop. The program used for analysis isEmpower by Waters. The user name should be “student” Password is needed—>just type “chem.421”6. The Project used for this analysis set is Chem421, and “chem421b” for system (The PC willtake ~ 3 min to connect to acquisition server)7 At the Empower main menu, select “Browse project and double click on “AAS method” whichwill mark the AA peak, and calculates the peak area of the AA peak. This method sets the parameters for the experiment......the flow rate should be 2.00 mL/min, the %A should be 80.0(which is doubly distilled water), %B should be 20.0 (which is HPLC grade methanol), %Cshould be 0.0, %D should be 0.0 The processing method integrate the peak of the chromatogramand marks the AA peaks. The report method generates a report from the processed data, and thisis printed.8. Click on the button with green arrow, the run button, to begin equilibrating the column. Theequilibration step will take 20 min. Once the column has finished equilibrating, the injectstandards” line should be in red, and the instrument status should say “waiting for injection”After you set program to equilibration step, you should begin to prepare your standards and other samples while waiting for the pressure to stabilize
Preparation of AA Standards
1. Prepare 5 mL of a stock 12 mM of 3 AA (Tyrosine, Tryptophan and Phenylalanine) standardsolution in mobile phase, by weighing out the appropriate amount of AA power and diluting withmobile phase in a 5 mL volumetric flask.2. Use the stock standard solutions to prepare the following mixed standards by dilution withmobile phase to prepare 1 mM, 2 mM, 3 mM, 4 mM in 5 mL volumetric flasks.
Running a Sample
1. With the 100
µ
L syringe located in the HPLC drawer, obtain 50
µ
L of each solution that youwould like to inject to HPLC system. Be sure that there are NO air bubbles in thesyringe before injection. Inject it into the HPLC while in the LOAD position. Theinjection port delivers only a 20
µ
L aliquot to the column: however a 50
µ
L excess is needed for complete loading. Next turn the valve on the injection port from the LOAD to the INJECT position. You will hear a beep that indicates an injection has occurred. Turn the valve back to theLOAD position after you heard the beep.2. You need to inject each standard and each sample three times. The computer willautomatically print out the chromatogram and the data after each run has been completed.3. Follow this procedure for all standards (1,2,3,4 mM), Vitamin pill sample and fresh fruit juiceor honey samples
Preparation of Vitamin pill Samples
of 00

Leave a Comment

You must be to leave a comment.
Submit
Characters: ...
johnson2232

Fruit Juice Versus The Whole Fruit http://j.mp/56iyZd

reply01 / 26 / 2010
kolita kamal

dear sir, i am kolita from nara, can u send me the amino acid detection method doc by HPLC to my mail kkamal@nara.ac.lk

reply06 / 09 / 2009
You must be to leave a comment.
Submit
Characters: ...