Small molecules are becoming more and more difficult to make, and macromolecules are developing very strongly. It seems to have become one of the important manifestations of the drug research and development market in recent years, and “peptide” drugs are one of the hottest topics. At present, the quality supervision of peptide drugs is becoming more and more strict. Under this trend, it is particularly important to find analytical methods that can meet higher resolution and sensitivity. Many small partners always encounter various problems in the development of peptide sample analysis methods, which are extremely annoying. Here I summarize some common problems in the development of peptide methods to share with you.

(Image source: soogif)

1.What is the difference between peptide content and peptide purity?

In addition to the polypeptide itself, a polypeptide product includes impurities such as water and organic salts brought in during the production process. The purity of the polypeptide only refers to the content of the peptide product and the content of impurities contained in the polypeptide itself, excluding impurities such as water; while the polypeptide content It refers to the net content of the target polypeptide in the product, which is generally detected by N element analysis or amino acid molecule methods; therefore, even if a polypeptide has a purity of 99%, because the product also contains water and organic salts, its content may be Also only 70-80%.

2.How to dissolve peptides?

Most peptides can be dissolved in ultrapure water. For some insoluble peptides, the amino acid sequence must be analyzed first. For acidic peptides, it can be dissolved in a small amount of alkaline (such as 0.1% ammonia) solution, and then diluted to the desired concentration. , For basic polypeptides, it can be dissolved in a small amount of acidic (such as acetic acid, trifluoroacetic acid) solution, and then diluted to the desired concentration. For hydrophobic polypeptides, it can be dissolved in organic solvents, such as DMF, methanol, propanol, isopropyl Alcohol, DMSO, etc.

3.Why should trifluoroacetic acid be added to the mobile phase as an ion pair reagent, and what other mobile phase systems or ion pair reagents can be used for the separation and purification of peptides?

The addition of trifluoroacetic acid can adjust the pH of the eluent, and at the same time act as an ion-pairing reagent to interact with the peptide, thereby enhancing the separation effect and significantly improving the peak shape.

Other mobile phase systems or ion-pairing reagents that can be used for the separation and purification of polypeptides include acetic acid system, phosphoric acid system, hydrochloric acid system, heptafluorobutyric acid, etc., which can achieve good separation effect by adjusting the pH appropriately.

In addition, the reason why trifluoroacetic acid is superior to other ion modifiers is that it is easy to volatilize and can be easily removed from the prepared samples. Interference is minimal.

4.What should I do if the column pressure of the chromatographic column for testing peptides increases and the column efficiency decreases?

Routine cleaning and maintenance of the chromatographic column can follow the factory instructions of the chromatographic column, but when analyzing protein samples such as peptides, a phenomenon is prone to occur: protein contamination. The main reason is that the packing at the head end of the column is clumped. If protein contamination occurs, it is recommended to use acetonitrile-water-trifluoroacetic acid = 50-50-0.1 small flow rate to backwash 60 times the column volume or backwash overnight.

5.Peptide samples have no peaks or unusually small peak areas on the new silica-based column, why?

This is because the non-specific adsorption sites on the fully porous spherical silica column produce dead adsorption to the peptide, resulting in no peaks.

The non-specific adsorption sites in the chromatographic column mainly come from residual silanol groups, residual heavy metals in silica gel, and silanol groups exposed after the bonding phase falls off, and the inner wall of the column is not passivated. These will have strong non-specific adsorption to peptide samples.

In fact, when starting to use a new chromatographic column, the non-specific adsorption of biological samples will be more serious, and the chromatographic column can be saturated with high-concentration samples. Pre-sample before the formal detection, so that the sample accumulates on the column and covers such a site, so that our future analysis will have a good chromatogram. It is recommended to inject samples every one minute, and continuously inject more than ten needles to cover the non-specific adsorption sites with excess sample. After the peak area and peak shape are stable, the sample can be formally detected.

6.Does TFA in the buffer only play the role of pH adjustment? The higher the concentration of TFA, the more severe the baseline drift. Does that mean that the lower the concentration of TFA, the better when the pH of the buffer allows?

(1) TFA plays a similar role as an ion pair. The general concentration is 0.05-0.1%. Too high a concentration will make the solution acidic. Long-term use may affect the life of the column.

(2) At the same time, TFA can inhibit the silanol groups on the surface of silica gel and improve the peak shape of basic compounds. Sometimes 0.1% TFA separation is not good. Consider increasing the concentration to 0.2%. But pay attention to rinse the chromatographic column in time after use.

7.What is the reason for the frequent baseline drift during peptide testing? How to deal with it?

Gradient elution with a fixed concentration of trifluoroacetic acid sometimes causes a shift in the absorption baseline at detection at 210-220 nm, which is the cause of baseline drift in many reversed-phase separations.

To reduce or eliminate the baseline drift caused by changes in the spectral absorption of TFA, it is necessary to make the detection wavelength as close to 215 nm as possible, and to add 15% less TFA in solvent B than in solvent A to compensate for the baseline drift. For example, when the trifluoroacetic acid in solvent A is 0.1%, 0.085% in solvent B can be used.

8.How to choose the column packing used in the purification process?

The physicochemical properties and hydrophobicity of peptides with different sequences are very different. In most cases, the molecular weight less than 4000 and hydrophilic peptides are best separated by C18 column, and the molecular weight is greater than 5000 and extremely hydrophobic peptides are best separated by C4 column. The C8 column is between the C18 and C4 columns, and its application effect is more similar to the C18 column; for some special selective peptides, the phenyl column can also be selected.

9.Also attached are common problems and solutions in the process of biological sample analysis

Common problem phenomenonReasonSolution suggestion
Column pressure rise• Foreign body blockage
• Impurities in mobile phase, sample
• Fragments of the piston seal
• Sample composition analysis
• Pre-filter the mobile phase and sample with a suitable filter
• Install online filters
• Clean lines, replace piston seals
• Prepare sample solutions using mobile phase
Chromatographic peak splitting, broadening, tailing• Improper piping connection resulting in dead volume
• Inappropriate mobile phase conditions
• Sample volume is too large
• Column deterioration
• Reconnect tubing
• Elution with mobile phase containing 0.1% TFA
• Reduced sample injection volume
• Confirm column performance with efficiency testing
Extended or unstable retention time• Fluid leakage
• Inappropriate mobile phase conditions
• Insufficient column equilibration time
• Check pump, piping series for leaks
• Use flow with 0.1% TFA
• Phase elution
• Well balanced
Shorter retention time• Use of strong acids or bases results in hydrolysis of the bonded phase
• Inappropriate mobile phase conditions
• Insufficient column equilibration time
• Check the pH of the mobile phase and sample
• Elution was performed with a mobile phase containing 0.1% TFA.
• well balanced

Welch materials has launched Welch biological sample analysis method development kit for peptide sample method development

Welch Biological Sample Analysis – Column Method Development Kit


Ultisil® XB-C18 (300Å),

Ultisil® LP-C18 (300Å),

Ultisil® XB-C4 (300Å),

Ultisil® XB-C8 (300Å),

Ultisil® LP-C8 (300Å);

Specifications: 4.6×250mm, 5μm

(Other specifications can also be selected).

★ Appropriate for method development of proteins, peptides or other macromolecules In order to better interact with the bonded phase, a large pore size (300Å) packing is required.

★Different selective bonding phases with different retention capabilities, meet the retention and separation of proteins and peptides of various molecular sizes.

Attached application case spectrum

Ethinyl estradiol impurity separation

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