Video Assignment CHM256- Chromatography

[00:00:05]Hello everyone. Before we start with the presentation, [music] let's get to know our group members. We have Doven Nadira, >> [music] >> Ivy Thomas Wong, Putri Zulaikha Amatahir, and Trivyaza Natasha Suwandi.

[00:00:17]Before we move further along into this video, [music] we need to understand what is chromatography. So, let's start with the basics.

[00:00:25]Chromatography is one of the most important techniques in analytical chemistry [music] as it helps us separate and identify substances within complex mixtures.

[00:00:34]>> [music] >> The process works by moving compounds between two phases. The first one is a stationary phase that stays fixed, and the other one is a mobile phase that flows through.

[00:00:44]Because [music] different compounds interact differently with these phases, they can be separated and studied individually. So, >> [music] >> in simple terms, chromatography is like giving each compound its own lane in a race and watching how fast or slow they move.

[00:00:58]>> [music] >> This makes this a powerful tool for ensuring accuracy, quality, and safety in science and industry.

[00:01:05]Now that we understand what chromatography is, [music] let's look at why this topic matters in everyday life.

[00:01:11]Next, we're moving on to why this topic matters.

[00:01:14]In our daily routines, [music] we often consume products such as sodas and energy drinks. These beverages usually [music] contain additives and stimulants. And one of the most common one is caffeine. [music] As people enjoy caffeine for its energizing effects.

[00:01:30]>> [music] >> But having too much caffeine can cause health problems such as insomnia, anxiety, [music] or even heart issues. This is where chromatography becomes more than just a classroom concept.

[00:01:41]By detecting caffeine in soft drinks, we connect [music] science directly to consumer safety.

[00:01:47]It shows how analytical chemistry protects [music] public health and ensures that product labels are transparent and trustworthy.

[00:01:53]>> [music] >> With this introduction now in place, we can move on to the theory behind caffeine detection and how chromatography makes it possible.

[00:02:03]>> [music] >> High performance liquid chromatography or HPLC is widely used in the food and beverages [music] industry to detect and measure the amount of caffeine in soft drink and energy drink. The main purpose of this application is to determine the exact concentration of caffeine in a beverages.

[00:02:21]The measured value is then compared [music] with the caffeine amount stated by the product label to ensure its accuracy. [music] This is important because food manufacturers must comply with food safety regulation and provide correct nutritional information to consumers. [music] Besides verifying label accuracy, HPLC also helps maintain food quality and consistency while preventing excessive caffeine levels that could cause health risk.

[00:02:50]The theory behind this application is based on the principle of high performance liquid chromatography. HPLC separates compounds [singing] according to how strongly [music] they interact with two phases, the stationary phase inside the chromatographic column and the liquid mobile phase that flows through the column under high pressure.

[00:03:11]After the beverage sample is [music] injected, the mobile phase carries all the components through the column. Since each compound interact differently with the stationary phase, they travel at different speeds. This allows caffeine to be separated from other ingredients such as sugar, preservative, coloring agent, and flavor compound.

[00:03:33]Once separated, caffeine pass through a UV visible detector because it absorb ultraviolet light at around 272 to 275 [music] nanometers.

[00:03:44]The detector produce a chromatogram. The retention time confirm [music] that the detected compound is caffeine while the peak area is proportional to its concentration, >> [music] >> finally, the peak area is compared with a calibration curve prepared from caffeine standards [music] to determine the exact caffeine concentration in the beverages.

[00:04:04]>> [music] >> Finally, let's take a look at the methodology and experiment result.

[00:04:09][music] First, let's look at the materials used to conduct this experiment.

[00:04:16]The soft drink samples were chosen [music] to be Coca-Cola, HPLC with UV detector, C18 column, 10 [music] mg of caffeine standard, methanol which HPLC grade, distilled water, >> [music] >> syringe filters, volumetric flask, and pipette.

[00:04:35]>> [music] >> Next, let's move on to the methodology.

[00:04:40]The process consists of six key steps.

[00:04:42][music] First, the soft drink sample was thoroughly degassed to remove [music] any dissolved carbon dioxide that could interfere with the HPLC system.

[00:04:52]Next, it was filtered using a 0.45 micrometer syringe [music] filter to eliminate any particulate matter.

[00:05:01]Third, the caffeine standard solution was prepared at a known concentration.

[00:05:06]For the analysis, 10 microliters of the standard and [music] 10 microliters of filtered sample were injected into the HPLC system. Once the separation took place, the sample peak was compared with the standard peak.

[00:05:21]Finally, the total caffeine concentration was calculated based on [music] these responses.

[00:05:30]>> Now, let's take a look at our results.

[00:05:33]As you can see from table one, the retention time for caffeine standard was 0.830 minutes, yielding a peak area of 56.617.

[00:05:43][music] For Coca-Cola sample, the caffeine peak appeared at a retention time of 1.307 minutes with a much larger peak area of 5358.417.

[00:05:59]To determine the exact concentration, we first calculated the response [music] factor of our standard as shown in the table two.

[00:06:08]>> [music] >> To calculate the response factor, we can divide the peak area of standard by concentration of standard.

[00:06:16]>> [music] >> RF equal to 56.617 divided [music] by 100 to get 0.56617 area per ppm.

[00:06:29]Next, we can use the response factor to find the amount of caffeine present in the sample.

[00:06:37]>> [music] >> Amount of caffeine can be calculated by dividing peak area of the sample by response [music] factor.

[00:06:44]Amount of caffeine equal to 5358.417 [music] divided by 0.56617 to get >> [music] >> 9467.99 ppm.

[00:06:58]This brings our final caffeine concentration in the Coca-Cola sample >> [music] >> to 9467.99 ppm.

[00:07:08]>> [music] >> Now, to conclude everything, through the analysis of the chromatograms, the caffeine standards, which is [music] 100 parts per million, eluted at a retention time of 0.830 minutes with a peak area of 56.617, [music] yielding a calculated response factor of 0.56617 area per parts per million. The Coca-Cola sample displayed a matching caffeine peak at a retention time of 1.307 minutes with a significantly larger >> [music] >> peak area of 5358.417.

[00:07:48]Now, by comparing the sample's peak area [music] against the standard's response factor, the final concentration of caffeine in the Coca-Cola sample was determined to be >> [music] >> 9467.99 parts per million or mg per L.

[00:08:04]Now, this methodology effectively demonstrate the precision of HPLC in food [music] safety and quality control, ensuring that consumers' products can be [music] accurately monitored for stimulant content to satisfy regulatory standards and protect public health.

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