Separation of Additives in Soft Drinks by HPLC using a Non-polar Column with Polar Endcapping is the title.
Microsoft Word - Analytical practicals Manual 17 Experiment 8: Separation of Additives in Soft Drinks by HPLC using a Non-polar Column with Polar Endcapping. The challenge of HPLC is to obtain retention of hydrophilic components in samples. The more commonly used C8 or C18 columns (reverse phase) do not always provide the selectivity necessary to retain such hydrophilic (polar) compounds in samples. Note that there is no such difficulty with the more non-polar components in a mixture. The introduction of HPLC columns containing polar endcapping and/or polar embedded groups allows for the retention of these hydrophilic compounds. The conditions described in this practical allow the separation of additives commonly found in soft drinks that cannot be separated on conventional alkyl-bonded phases (C18). The major problem in analysing soft-drink additives is retaining the highly polar components while keeping the retention of the hydrophobic analytes down to an acceptable k’ range. Because of its highly polar selectivity, ascorbic acid is virtually unretained using C18 or C8 columns when using conditions that elute the more hydrophobic components within a working k’ range. Using the Synergi Polar RP column the ascorbic acid is retained beyond the void volume while the last eluted peak has a k’ value of approx. 5. Column: Synergi 4μm Polar-RP 80A (150 x 4.6 mm) Synergi is based on a new 80A ultra-high purity (> 99.9% metal free), base deactivated type silica, which ensures minimal surface metal sites available for chelation and reduced silanol (Si-OH) acidity even at neutral pH. The high metal content of older type silicas can result in poor recovery or adsorption of certain analytes due to chelation and contribute to peak tailing of basic compounds. 4μm generates columns with efficiency intermediate between 3 or 5μm and with a pressure drops similar to those obtained with columns packed with 5μm particles. (unexpected – should be higher – this unexpected bonus is due to the different type silica) 80A Pore Size for High Surface Area: The high (475m2/g) surface area results from a unique 80A pore geometry. This increases sample analyte interaction with the bonded phase and it also compensates for the reduced hydrophobicity of Synergi 4μ Polar-RP, allowing it to retain both hydrophobic and hydrophilic analytes through non-polar and polar interactions respectively. Synergi-Polar-RP: Is an ether linked phenyl phase with proprietary hydrophilic endcapping designed specifically to maximise retention and selectivity for polar, aromatic analytes that are so often encountered in the pharmaceutical industry Due to its bonding chemistry, Syn. Polar-RP displays a polar selectivity that complements the more conventional selectivity of Syn. Max-RP. The ether – linkage not only improves selectivity for acidic and basic compounds but also gives high aqueous mobile phase stability. Eg. Methanoic acid can be separated on an embedded basic group (N - containing amide link) on a non-polar column. The basic group however can interfere with the resolution of highly , polar compounds. However since the SP used here uses an ether linkage as the embedded polar group, the result is improved peak shape and separation of highly acidic, polar analytes. In addition this SP’s ether linkage is 18 extremely resistant to hydrolysis even at pH 1.5, thus enabling separations under relatively harsh conditions. At the other end of the pH spectrum, it is stable to pH 7.0. Mobile Phase 55 of .02M KH2PO4 adjusted to pH 2.3 then mixed with: 45 of Methanol @ a flow rate of 1.0cm3/min. Detector: UV detector at 220nm. (Preferably Diode Array). Procedure: 1. Stock solutions of the following standards are provided with concentrations of 100mg/100cm3: Ascorbic Acid; Acesulfame K; Saccharin; Aspartame; Sorbate; Benzoate; Caffeine. The compounds elute in this order from the RP-Polar column.. 2. Using the stocks provided make up standards of concentration 1, 2, 3, 4, & 5 mg/100cm3 in 100cm3 volumetric flasks. Use the mobile phase as solvent. One Exception: Take 5, 10, 15, 20 & 25cm3 of the Ascorbic acid solution. 3. Samples: Take 25cm3 of each of three drinks and dilute to 100cm3 with MP. Important Note: If you do not obtain a peak for aspartame in Lilt and Energise Sport you will have to inject undiluted samples of these to measure the amount of aspartame in these drinks. 4. Analyse each of the 8 solutions on the RP-Polar column and determine the % of each component in each of the three drinks. (Can be 10 injections – See 3 above) 5. Meanwhile take 1.0cm3 (approx) of each of the stocks and dilute each one separately to 10cm3in eight 10cm3 volumetric flasks. Analyse these on a 150mm C18 column on a separate HPLC using the same mobile phase. Report their retention times and compare with your results from the other HPLC. Compound tr on RP-Polar using 45:55 Methanol:KH2PO4 tr on C18 using 45:55 Methanol:KH2PO4 Ascorbic Acid Acesulfame K Saccharin Aspartame Sorbate Benzoate Caffeine 19 6. Draw the appropriate graphs and determine the % of each component in each drink. Ascorbic Acid ____%; Acesulfame ____%; Saccharin ____%; Aspartame ____%; Sorbate ____%; Benzoate ____%; Caffeine____%; 7. Look up the structures of each component and rationalise the tr values with the structure, the mobile phase used and the stationary phase. 8. Comment on the difference between the tr values under the two sets of experimental conditions. Could the C18 column and its mobile phase be used? Give the reasons for the differences between the two sets of tr values. 9. Read the article “The Benefits of Reversed Phases with Extended Polar Selectivity in Analysing Wide-Polarity – Range Samples” in LC-GC March 2002 pages 156 –164. This will help you to answer the above questions. Synergi Polar Reverse Phase Column: Increased retention of highly polar and aromatic compounds Highly reproducible and stable phenyl phase Enhanced polar resolution in 100 % buffer mobile phases Peak area STDs mg/100cm3 STD 1 STD 2 STD 3 STD 4 STD 5 Ascorbic acid 14737 29693 45611 58791 72285 Acesulfame K 8175 15862 24128 32109 40033 Saccahin 9640 20168 30633 40020 49831 Aspartame 1931 3809 5883 7853 9818 Sorbate 3369 6700 10056 13180 16461 Benzoate 13713 28094 43145 56549 73874 Caffeine 18692 37482 56515 76383 95486 Energise sport Lilt Red bull Ascorbic acid 24324 Ascorbic acid 3235 Ascorbic acid 3235 Acesulfame K 20544 Acesulfame K 1515 Acesulfame K 1515 Saccahin not detected Saccahin 928 Saccahin not detected Aspartame 1860 Aspartame 2270 Aspartame 2270 Sorbate 17363 Sorbate 2016 Sorbate not detected Benzoate 55001 Benzoate 6434 Benzoate 2029 Caffeine not detected Caffeine not detected Caffeine 236116 LC•GC Europe December 20022 COLUMN WATCH Reversed-phase chromatography is by far the most widely used mode in high performance liquid chromatography (HPLC).1 It allows chromatographers to manipulate the mobile phase by changing organic solvent type, solvent composition and pH; by adding modifiers such as surfactants, chiral reagents, competing bases and ion-pair reagents; or by adjusting experimental conditions such as flow-rate and temperature. Originally, researchers believed that they would need only a few stationary phases to achieve virtually any separation they encountered. Indeed with a C18 column and the ability to adjust the myriad parameters, chromatographers have achieved many successful separations. Still, sometimes analysts can neither find the necessary selectivity nor obtain a rugged and reproducible separation easily, no matter how many parameters they adjust. Early Modified Surfaces in Reversed-Phase Chromatography New and modified reversed-phase chromatography stationary phases have been introduced throughout the years to provide more separation power. In reversed-phase chromatography, basic compounds can frequently interact with unreacted silanols on the silica gel because it is virtually impossible, at least with monomeric bonding, to remove or cover all of the silanols because of steric reasons, especially with long alkyl chain phases such as octadecylsilane (C18). This interaction is most problematic when the packing is used at intermediate pH values of pH 4–8 at which silanols and many basic compounds are partially ionized. Twenty years ago, the low-purity silica gel used for most HPLC columns caused high surface acidity and a tailing problem with basic compounds. The first modified stationary phases were the so-called base- deactivated bonded silicas. These base- deactivated phases were not deactivated with base, but the term implied that the column had been treated to provide minimal interaction and tailing with strongly basic compounds