QUANTIFICATION OF CAFFEINE AND BENZOIC ACID BY HPLC-RID

Write a pre-lab report using the following information: 

(a) You are given two stock solutions, each one containing one of your two analytes at a known concentration of 1,000 mg/L in water. From these stock solutions, make mixed calibration standard solutions containing 5, 10 or 20 mg/L of each analyte in the same solvent, using variable micropipetters with a working range of 100 – 1,000 µL volume, and 10 mL volumetric flasks. Also, prepare a calibration blank using the same solvent.

You are given an unknown sample, containing both DOP and DNP in unknown (to you) concentrations between 50 and 200 mg/L. Diluted this sample appropriately prior to analysis! Perform the appropriate dilution, and then split this sample into three aliquots of equal volume. The first two will be run as is as duplicate samples. Spike the third aliquot with the two analytes at a concentration corresponding to 100 mg/L in the original sample. Ideally, one would actually spike the original sample prior to dilution, but since that would be more complicated in this case, and one wouldn’t expect analytical problems during sample dilution (other than human error), we perform dilution and spiking in one step here.

Label all of your samples properly (name, date, content of container), and store them in the refrigerator until the day of analysis.

Measurement and analysis of samples 

 Analyze your samples by GC-MS in the following order:

Calibration blank

Calibration standard 5 mg/L 

Calibration standard 10 mg/L

Calibration standard 20 mg/L 

Unknown sample (diluted appropriately)

Unknown sample duplicate (diluted appropriately)

Unknown sample (diluted appropriately) + spike of 100 mg/L (in the original sample) of each analyte

Continuing calibration verification (CCV) sample (= calibration standard 10 mg/L)

(b) Explain step-by-step how you will use the chromatograms of the calibration solutions (including the calibration blank) and of the unknown sample to determine the concentration of the analytes in your unknown sample. 

(c) Explain in detail (i.e. what exactly you will do, and how) the proper way of making a hundred-fold dilution of a stock solution in the same solvent, using a variable micropipetter (working range 100 – 1,000 µL) and a 100 mL volumetric flask. 

 Introduction
Chromatographic techniques are used in science areas since they enable the scientists to single out and determine the constituents of a sample. Frequently, high pressure liquid chromatography with refractive index detection (HPLC-RID) is employed in identification of xylene isomers in most products (Kromidas, 2008). In this experiment, high-performance liquid chromatography (HPLC-RID) is used to quantify the percentage of caffeine and benzoic acid in the solution. 

Chromatographic methods are based on a simple principle that analytes in a sample are identified based on their preferences for one of the solvents (phase) to the column (small-diameter packed with small particles) (Xiu-Qin et al., 2009). For instance, if a nonpolar solvent and a polar solvent are in contact, polar molecules will interact with polar solvent, and vice versa in the case of nonpolar molecules. Usually, in chromatography, the column is always a stationary phase while the solvent is the mobile phase which flows over the column.

Preparation of the solutions 

Calculations for calibration standards (part A)

(i)                 5mg/L mixed calibration standard solution

Let V1 to be unknown volume (?), V2= 10mls, C2= 5mg/L, C1= 1000mg/L, where V= volume while C= concentration of the samples.

V1= (10mls X 5mg/L)/ (1000mg/L)

= 0.05mls, (0.05ml X 2 = 0.1ml for both stock solutions)           

(ii)               10mg/L mixed calibration standard solution

V1=? V2= 10mls, C2= 10mg/L, C1= 1000mg/L

V1= (10ml X 10mg/L)/ (1000mg/L)

= 0.1ml, 0.1ml X 2 = 0.2mls

(iii)             20mg/L mixed standard calibration solution

V1 =? V2= 10mls, C2= 20mg/L, C1= 1000mg/L

V1= (10mls X 20mg/L)/ (1000mg/L)

= 0.2ml, (0.2ml X 2) = 0.4mls

(iv)             Blank solution from the stock solutions 

V1=? V2= 10ml, C2=1000mg/L, C1=1000mg/L

V1= (10ml X 1000mg/L)/ (1000mg/L)

= 1ml, (1 X 2) = 2mls

(v)               Unknown(both containing DOP and DNP) of concentration between 50mg/L and 200mg/L

Dilute the sample as follows: take 2ml of the solution, place it in 10ml volumetric flask and top up to the mark with distilled water and divide it in the three aliquots of each 3.3mls each.

(vi)             100mg/L concentration of the analytes(in the original sample stock solutions)

V1=? V2= 10ml, C2= 100mg/L, C1= 1000mg/L

V1= (10nl X 100mg/L)/ (1000mg/L)

= 1ml (1ml X 2) = 2mls (for both stock solutions)

Dilutions of the solutions (Part B)

(i)                 Measure 0.05ml of each stock solution using micropipetter into 10ml volumetric flask to make mixed standard solution (5mg/L). Top up to the mark with distilled water and label with it as 5mg/L mixed standard calibration solution and close its top.

(ii)               Repeat the same procedure in (i) but now using 0.1ml of each stock solution and labeling it as 10mg/L mixed standard solution.

(iii)             Pipette 0.2ml of each stock solution into 10ml volumetric flask. Top up to the mark with distilled; close the top and label precisely as 20mg/L mixed standard solution.

(iv)             Measure 1ml of each stock solution, and then pour it in 10ml volumetric flask, top up to the mark with distilled water, close the top and mark it appropriately as the blank of mixed standard solution.

(v)               Follow the steps as described in (v) Part A above for dilution. For the two (duplicate samples) top up to the mark with distilled water while the last aliquot spike it by adding 6.7mls of 100mg/L concentration (analytes) as prepared in (iv) part A and mark it accordingly as spiked sample while the other two as duplicates samples. Store all samples in refrigerator for analysis.  

 Measurement and analysis of samples 

Ensure all settings of the MS-GC are working properly. Rinse and fill the syringe with working solution starting  with calibration blank, calibration standard 5 mg/L, calibration standard 10 mg/L, calibration standard 20 mg/L, unknown sample, unknown sample duplicate, unknown sample (diluted appropriately) + spike of 100 mg/L (in the original sample) of each analyte and finally continuing calibration verification (CCV) sample (= calibration standard 10 mg/L). Place 0.10mls of the solution through the septum while not removing the syringe. Adjust injector in inject position and release the sample onto the column by immediately pressing the start button on computer. Three peaks will appear on screen then stop it after 60 seconds and when the third peak has returned to baseline. Convert this time in minutes and ensure that time is set per chromatogram (Robinson, Skelly & Frame, 2014). Save it and repeat the procedure for the other solutions. 

At the end, the following results are expected: chromatograms of each solution, preparation of calibration peaks versus concentration of each sample, concentration of concentration of analytes and retention times of solutions.

Identification of concentration of analytes in unknown solution using chromatograms of calibration of solutions

First, make a calibration peak area of the plot at 280nm vs. caffeine concentration which contains point (0, 0) to represent blank solution signal then obtain the correlation coefficient (R²). After this, make calibration peak area plot at 240nm vs. concentration of benzoic acid.  Indicate point (0, 0) to denote blank solution. Get the equation of the correlation coefficient (R²). In the chromatograms of the solutions, use the retention times of the samples to identify the unknown solution and then the calibration plot is used to determine the concentration of analytes (mg/L). The caffeine and benzoic acid peaks in the chromatograms of soft drink and unknown samples.  Use the calibration plot to determine the caffeine and benzoic acid concentrations (mg/L) for the soft drink and the unknown solution.

How to prepare hundred-fold stock solution in the same solvent 

To make hundred-fold dilution of a stock solution in the same solvent, one should determine concentration of each stock solution that will occur in 100ml volume. The obtained volume is then pipetted from each stock solution into 100ml volumetric flask, which is then topped up to the mark with distilled water

References

Kromidas, S. (2008). More practical problem solving in HPLC. Hoboken, NJ: John Wiley & Sons.

Robinson, J.W., Skelly, E.M. & Frame, G.M. (2014) Undergraduate instrumental analysis. Boca Raton, FL: CRC Press.

Xiu-Qin, L., Chao, J., Yan-Yan, S., Min-Li, Y., & Xiao-Gang, C. (2009). Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chemistry, 113(2), 692-700.