Gc 1

lIntroduction to High-Performance Liquid Chromatography (HPLC)

injection volume, we make peaks smallerTranscript

0:00

It gives you a good lot of that information, but it's not gonna tell you about the fact that if there there is the ability to use dual blade like a walker. You could comment on the fact that you haven't assessed the lambda max for either of your APIs. So let's say that theobromine and caffeine are our APIs. Yeah. You could say we haven't done a scan of those, and perhaps we could employ the dual wavelength detector to better quantify each of them.

1:57

Because, say, for example, we're running this at two fifty four nanometers. Yeah. If caffeine was best at two seventy five or two seventy, whatever it is, the t o bromine might absorb best at two twenty. Yeah. It might be a case of two fifty as just a a middle ground.

2:13

Okay. But it may be better to run the two of them separately. Okay. Because you might not get any absorbance of caffeine at two twenty, you and might not get any absorbance of theobromine at two seventy. And you could effectively get one peak on each chromatogram then.

2:26

And you're more confident then that you're analyzing only the right thing. Does that make sense? The further apart those wavelengths go, the more confidence you would have in that. Yeah. Is that okay?

2:37

Yeah. But it it generates less data for each peak, if that makes sense. The dual wavelength? The dual wavelength because it's Okay. Resolution or enough resolution.

2:55

Get less in a pixels. You mentioned a TV. There's just less information in it because it's not taking them every split second. It takes on every other split second. Okay.

3:05

Does that make sense? Then the needle will draw your solution up. What's on the screen now there is the solvent front. So it's just the first part of your injection that hits the detector. That's gonna go to nothing nanosecond whenever your peaks start to come out.

3:41

So this is your peak starting there. So you can see there that your solvent front pretty much disappears. Again, that's just based on your scale. So these are our starting conditions with 40% water, 60% methanol. And our pigs are very close to coming out on top of each other.

4:15

So we need to change things. So we're gonna change the gradient. But unlike in GC where we would have run mixture only mixture until we had conditions we liked. With HPLC, there's a a lot longer time needed between injections under different conditions because the whole system has to equilibrate. So we're gonna do the three injections of each at each stage that we change our conditions.

4:46

And what base condition is this? This is 40% water, 60%. And you said you were using dual something again? No. No.

5:14

We're using a single wavelength to set up. Single wavelength. What you do, you have the ability to set up the dual wavelength, and it'll generate two chromatograms for each drug we do. So that's our first peak. This is what's gonna be on your this is your report.

5:36

So you'll see What your sample name is is up here. So in case you're wondering what the injection is, you'll see it there. It'll be that's more important when it comes to the single injections because it'll help you identify what the peaks are. Okay? And the mixture year, you're gonna get to do your injection date and time will help you with figuring out what we ran when.

6:00

You don't really need to worry about anything else here, maybe the injection volume. If we reduce our injection volume, we make peaks smaller. Smaller peaks will be less wide. So if they are sort of trying to overlap, which you can if you do a really big injection, I actually might do an injection of about 10 or 15 microliters just to see that that resolution will will get worse. So our resolution is down here.

6:29

It's 1.93. So you typically would want your resolution to be above two. And then your peak tailing factor is 1.24, 1.23. That's acceptable. And the monographs will guide you towards or having a peak tailing factor between zero point eight and one point five.

6:55

Okay. So if it's less than one, that means that if your peak is fronted. And if it's greater than one, that means your peak is tailed. So about about 1.2 is

  • The role of HPLC in analyzing active pharmaceutical ingredients (APIs)

  • Focus on the specific example of caffeine and theobromine as APIs

Dual Wavelength Detection in HPLC

  • Importance of assessing the lambda max for each API

    • Lambda max: Wavelength at which a substance shows maximum absorbance

    • Specific case:

    • Caffeine may have optimal absorption at wavelengths around 270-275 nm

    • Theobromine may absorb best around 220 nm

    • When using a single wavelength (e.g., 254 nm), results may be suboptimal because:

      • Caffeine may not absorb well at 220 nm

      • Theobromine may not absorb well at 275 nm

  • Benefit of dual wavelength detection:

    • Ability to generate individual chromatograms for each API, increasing specificity

    • Greater confidence in results due to clearer identification of peaks for each compound

Potential Issues with Single Wavelength Detection

  • Using a single wavelength can lead to:

    • Overlapping peaks which complicate analysis

    • Potential misidentification of the compounds present in the mixture

  • Resolution consideration:

    • Using different wavelengths for each API provides clearer, separated peaks allowing accurate quantification

Data Generation and Peak Analysis

  • Explanation of solvent front behavior during injection:

    • The leading portion of the sample entering the detector is referred to as the solvent front

    • After the solvent front, the actual peaks appear, starting with the first peak after the front

  • Injection settings:

    • Starting conditions set at 40% water and 60% methanol

    • Mention of similar peaks appearing closely together indicating potential overlap

Injection Protocols in HPLC

  • Importance of equilibrating conditions between different injections:

    • Unlike Gas Chromatography (GC), HPLC requires more time for the system to stabilize after condition changes

    • Perform three injections at each condition stage for reliable data

  • Consideration of injection volume:

    • Smaller injection volumes lead to smaller and narrower peaks, aiding in resolution

    • Larger injection volumes may result in overlapping peaks

    • Recommended injection volume: 10 to 15 microliters for better resolution

Analysis of Chromatogram

  • Following parameters are critical:

    • Resolution: The value recorded is 1.93, optimum threshold above 2

    • Peak tailing factor: Recorded as 1.23, acceptable range is between 0.8 and 1.5

    • A peak tailing factor below 1 indicates peak fronting

    • A factor above 1 indicates peak tailing

Sample Analysis and Presentation

  • Identification of components:

    • In the provided analysis, caffeine is indicated by a peak at 1.91 minutes and theobromine at 1.7 minutes

  • Ensuring clarity in reporting:

    • Importance of including chromatograms in presentations

    • Details of peak characteristics (tailing factors, resolution) included in the report

    • Mobile phase composition and acquisition method are essential to mention

Conclusion

  • Overall methodology emphasizes the importance of meticulous setup in HPLC for accurate quantification of APIs

  • The dual wavelength approach and careful analysis of peaks enhances the reliability of chromatographic data results for caffeine and theobromine analysis.