During discussions about the puriFlash® MS, the maximum mass limit of this detector (2000 m/z for the puriFlash® MS L) often comes up. This is particularly relevant when dealing with high molecular weight compounds, such as large peptides. To speed up the identification of these compounds, our system includes an algorithm called charge deconvolution, which helps determine the exact molecular mass.
Let’s walk through how this works using an example application. We’ll demonstrate just how straightforward it is to obtain this information.
### Charge Deconvolution: Simplifying Signal Intensity
In mass spectrometry, peptides typically appear with an ESI source. Under "soft" ionization conditions, they can become multiply charged, making them visible across the mass range of the puriFlash® MS. By knowing the charge states of the peaks in the mass spectrum, an algorithm can automatically calculate the original molecular mass of the peptide.
The deconvolution algorithm used in Advion combines predictive charge envelope generation with overlap calculations using actual MS data. It applies a maximum entropy goodness-of-fit approach and removes artifacts by determining peak significance. This innovative method allows for quick deconvolution over a broad mass range within seconds. It also outputs neutral masses, where the signal intensity is directly linked to the MS raw data intensity and the analyte concentration in solution.
In practice, performing this operation on the puriFlash® MS is quite simple:
1. **Acquire the Mass Spectrum**: Use the "Advion Data Express" software to capture the mass spectrum of the compound.
2. **Remove Background Noise**: For better visualization, clean up the signal by removing background noise.
3. **Perform Charge Deconvolution**: Click on the deconvolution button to begin the process.
When you click on the "Deconvolve" button, a window opens where you can specify parameters like the input and output mass ranges, the charge range, and any adduct masses. After clicking "OK," the deconvolved spectrum will display.
### Application Example
To illustrate this, we purified four peptides with molecular weights ranging from 10 to 27 kDa using reverse-phase chromatography. Since we didn’t know the elution order of the peptides, they were collected and then identified via direct injection into the puriFlash® MS. Our goal was to optimize the method to shorten the run time.
**System Specifications:**
- puriFlash® 5.250P
- Solvents: A = Water + 0.1% AF, B = Acetonitrile + 0.1% AF
- Column: PM-30C18-F0025
- Flow Rate: 15 mL/min
- Injection Mode: Liquid (lyophilized compounds reconstituted and dissolved)
- Injected Volume: 150 µL
- Compounds:
- Cytochrome c from horse heart (P/N: 1J484A)
- Lysozyme from chicken egg white, salt-free (P/N: 6D6694)
- Myoglobin Type I from horse skeletal muscle (P/N: 294440)
- Chymotrypsinogen A from bovine pancreas (P/N: IYQ650)
**Gradient:**
| Time (min) | A (%) | B (%) |
|------------|-------|-------|
| 00:00 | 75.0 | 25.0 |
| 23:00 | 47.0 | 53.0 |
| 23:03 | 0.00 | 100 |
| 30:00 | 0.00 | 100 |
**Results:**
After purification and analysis, the results were as follows:
1. **Peak 1:** Identified as Cytochrome C with a molecular weight of ~11.8 kDa.
2. **Peak 2:** Identified as Lysozyme with a molecular weight of ~14.3 kDa.
3. **Peak 3:** Identified as Myoglobin with a molecular weight of ~17 kDa.
4. **Peak 4:** Identified as Chymotrypsinogen with a molecular weight of ~25.6 kDa.
Following identification, the purification method was optimized to reduce the runtime by 60% using puriFlash® Monolith columns.
**Optimized Gradient:**
| Time (min) | A (%) | B (%) |
|------------|-------|-------|
| 00:00 | 75.0 | 25.0 |
| 07:00 | 47.0 | 53.0 |
| 07:03 | 0.00 | 100 |
| 11:00 | 0.00 | 100 |
This optimization significantly reduced the purification time while maintaining the desired purity levels.
### Conclusion
The puriFlash® MS not only accelerates compound identification but also enhances purification efficiency. Combined with the performance of puriFlash® Monolith columns, it offers a powerful tool for researchers. Additionally, the puriFlash® MS is highly versatile, capable of coupling with HPLC systems, analyzing TLC spots, and identifying large molecules through charge deconvolution.
For further details, visit [www.flash-chromatographie.com](http://www.flash-chromatographie.com) or feel free to reach out to us with any questions!
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