> For the complete documentation index, see [llms.txt](https://docs.rplpeptides.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://docs.rplpeptides.com/docs/faq/hplc-faq.md).

# HPLC FAQ

## 1. What HPLC method does RPL Peptide use?

RPL Peptide uses reversed-phase HPLC (RP-HPLC) with C18 stationary phase columns (typically 4.6 × 250 mm, 5 μm particle size). The mobile phase consists of water/acetonitrile gradients with 0.1% TFA as an ion-pairing agent. Detection is performed at 214 nm (peptide bond absorption) and 254 nm (aromatic amino acid absorption).

## 2. What is the typical gradient for peptide analysis?

A representative analytical gradient: 5%→65% acetonitrile (with 0.1% TFA) over 30 minutes at 1.0 mL/min flow rate. Gradient conditions are optimized per peptide sequence to achieve baseline separation of the target peak from impurities.

## 3. How do you identify impurities in the HPLC trace?

Common impurities include: deletion sequences (missing one or more amino acids), truncation fragments (incomplete chain assembly), oxidation products (Met → Met sulfoxide), and dimer/aggregate peaks. Identity is confirmed by LC-MS analysis of individual peaks.

## 4. What are acceptable system suitability parameters?

| Parameter                            | Acceptance Criteria         |
| ------------------------------------ | --------------------------- |
| Theoretical plates (N)               | > 2000                      |
| Tailing factor (T)                   | 0.8 – 1.5                   |
| Resolution (Rs)                      | > 1.5 from nearest impurity |
| % RSD (retention time, 6 injections) | < 1.0%                      |
| % RSD (peak area, 6 injections)      | < 2.0%                      |

## 5. Why is detection at 214 nm preferred over 254 nm?

Peptide bonds absorb strongly at 214 nm, providing approximately 10× greater sensitivity than 254 nm detection (which only detects aromatic residues). Purity reporting is based on 214 nm data; 254 nm data serves as supplementary information.

## 6. Can you provide raw HPLC data files?

Raw HPLC data files (in .txt or .csv format) can be provided upon request for qualified research institutions. Please contact technical support with your specific requirements.

***

*For technical HPLC inquiries: <technical@rplpeptides.com>*


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