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Educational - HPLC

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On this home page, I'll introduce my Tecnical Teory  and  Project in  my  daily Work. 




The main purpose of this experiment is to use the method of external standard to determine the composition of a sample solution.

Quantitative analysis with HPLC is similar to GC. We must identify and quantify one or more components from a complex mixture. The first challenge is to isolate the desire unknown and after identified we must quantify it. In HPLC the identification information depends only on retention time data. Under a given set of HPLC conditions, namely the mobile- and stationary-phase compositions, the mobile-phase flow rate, the instrument dead volume, the retention time is a particular value for each component. It changes only when one of the above parameters changes.
 The reproducibility of the amount injected is not nearly the problem with HPLC. In general is injected (5 –20 ?L), and there is not loss during the injection since the sample is not loaded into a higher-pressure system through a septum. In addition, the sample loop is manufactured to have a particular volume and is often the means by which a consistent amount is injected, which means reproducibility is maximized through the consistent “overfilling” of the loop via the injection syringe. In this way, the loop is assured of being filled at each injection and a reproducible volume is always introduced.
 Standard is anything taken as a basis of comparison. In this case we use an external standard to determine the composition of sample solution. External is an adjective that means on the outside or to be used outside of the body. In this case the external is for an external standard that is used out of the sample to form a standard curve. The standard curve is a graph showing the response of an analytical technique to know quantities of analyte.
 The duplication has an important rule because we need to determine the peak areas given by a range of standards and plot the peak area versus concentration of the standards injected. After that we can then determine the unknown concentration from a graph. The graph can be done by spot calibration, which consists of some concentration of a determined number of standards at different concentrations or by full calibration, which consist of a great number of standards at different concentrations to give more accurate calibration curve.

Materials and System Set-up:


Column: C18, 15 cm x 4.6 mm, 5 ?m.
Detection: UV 254 nm

External Standards: 10, 25, 50, 75, 100 ?g/ml each acetaminophen and caffeine respectively for external standards.
Unknowns: A or B containing unknown amounts of acetaminophen and/ or caffeine.
Flow rate: 1.5 ml/min.
Mobile phase: 15% CAN: 85% Water.
Detector SInjection Volume: 20 ?l
ens: 1.0 AUFS.



 In this method we prepare a standard containing the compound or compounds that we wish to determine, ideally present at about the same level as in the unknown, and we compare the chromatograms of standard and unknown. From the standard chromatogram, a response factor giving us the concentration of component that produces unit detector response:
 Response factor = concentration of component
         Peak height of area
 Then for the unknown chromatogram we can calculate the concentration of each component of interest by multiplying the peak height or area by the appropriate response factor.
 For this method to work, the detector response must be linear for each compound over the range of concentration being used. And also we have to inject exactly the same amount of material for each of the two chromatograms, so successful operation of the method depends on our being able to inject samples with good precision.
 The standards were prepared by the following way:
 Stock standard concentration was 2 mg/ml. It was mixed 20 ml from the stock solution of acetaminophen with 20 ml from the stock solution of caffeine to give a concentration of 1 mg/ml.
 2 mg/ml x 20 ml/40 ml = 1 mg/ml.
 From the above solution were pipetting the standards to make up a table known concentration standard solution for the external calibration curve.

Table # 1. Standard Concentration Solutions.
Concentration Acetaminophen 1 mg/ml Caffeine 1 mg/ml
10 ?g/ml Dilute 1 ml to 100ml in a Volumetric Flask. Dilute 1 ml to 100 ml in a Volumetric Flask.
25 ?g/ml Dilute 2.5 ml to 100ml in a Volumetric Flask. Dilute 2.5 ml to 100ml in a Volumetric Flask.
50 ?g/ml Dilute 5.0 ml to 100ml in a Volumetric Flask. Dilute 2.5 ml to 100ml in a Volumetric Flask.
75 ?g/ml Dilute 7.5 ml to 100ml in a Volumetric Flask. Dilute 7.5 ml to 100ml in a Volumetric Flask.
100 ?g/ml Dilute 5 ml to 50ml in a Volumetric Flask. Dilute 5 ml to 50ml in a Volumetric Flask
 The samples were previous prepared by the technician and ready to use. However, the outline from the experiment describe the preparation as follow:
 Stock sample solution: Weigh 20 tablets then ground into fine powder, weigh a portion which equivalent to one tablet weight to a volumetric flask (volume depends on what sample you have). Half fill with acetonitrile, sonicate for 30 minutes, then dilute to volume with acetonitrile.
 Working sample solution: Pipette certain volume (eg. 1,0 ml) of stock sample solution into a 10 ml Volumetric Flask, dilute to volume with mobile phase. Filter through 0.45 ?m filter into vial. Duplicate injections for each sample.
 The unknown samples were labeled by numbers, sample 1, sample 2; sample 3 and sample 4 and their supposed composition are acetaminophen and caffeine.
 The raw data recorded was the peak area of each chromatogram.
 The peaks were identified by the retention time, which was obtained by standards in the same conditions of the sample injection.



Part A – Raw Data:

Table #2.

 Acetaminophen (peak area) Caffeine (peak area)
10 ?g/ml 361.33 159.84
25 ?g/ml 885.68 390.29
50 ?g/ml 1731.63 791.09
75 ?g/ml 2582.29 1169.00
100 ?g/ml 3425.02 1545.87
Sample 1 1372.18 1244.17
Sample 2 1374.26 1244.21
Sample 3 2790.38 648.03
Sample 4 2786.20 637.08


Part B – Calculated Results:

 The standard curves for acetaminophen and caffeine was plotted by Excel Software see attached.

 The linear regression, which best fit the Excel Software did the concentrations of acetaminophen and caffeine, please see the graph attached.

 The concentrations of acetaminophen and caffeine in the unknown samples were calculated by the graph of standards of respective substances.

Table #3. Concentration of Acetaminophen and Caffeine in the Unknown Samples Obtained from Standard Curve for Acetaminophen and Caffeine.

 Acetaminophen (?g/ml) Caffeine (?g/ml)
Sample 1 38 79
Sample 2 39 79
Sample 3 80 40
Sample 4 79 39