HomeLarge Molecule HPLCOptimising the LC-MS Analysis of Biomolecules

Optimising the LC-MS Analysis of Biomolecules

Rudolf Köhling

AnalytiX Volume 8 Issue 2

Columns, solvent blends and calibration standards to maximise performance of LC-MS analyses

Mass spectrometry (MS) is an invaluable tool for the analysis of biomolecules like peptides, proteins and structural components, and small molecules like metabolites, lipids, steroids, sugars, amino acids, nucleotides and many others. A combination of a sensitive mass spectrometer and an HPLC system fitted with a suitable column and mobile phase can guarantee a successful analysis of complex biological samples. Sigma-Aldrich offers analysts a comprehensive line of premier MS consumables, including HPLC columns, high-purity solvents, additives and customised solvent blends and standards for calibration and troubleshooting to get the highest sensitivity, precision and resolution out of your HPLC and MS systems.

Optimising the HPLC column

To optimise the column selection for a reversed-phase separation of biomolecules, there are several important factors to consider. First, the pore size of the support particle must be large enough to permit access to the surface area inside the pores. Generally, it is recommended to use a wide pore particle, 300 Å or higher, for peptides above 30 amino acid residues (~3,000 Da). Second, activity of the support particle should be taken into account. Interactions should be limited to those between the analytes and stationary phase, avoiding relatively strong interactions like hydrogen bonding, chelation or ion exchange. This is especially acute with basic compounds, like peptides rich in ARG, HIS or LYS residues. Third, the chemistry of the bonded phase should be chosen to give the desired retention and selectivity.

For peptides below 3,000 Da and other small molecules, Ascentis® HPLC phases offer analysts a highly inert surface for excellent peak shape coupled with the benefit of choices in stationary phase selectivity. High-speed, high-resolution separations are also possible using Ascentis Express. For larger peptides and proteins, silica-based Discovery® BIO Wide Pore phases offer inertness and the necessary pore size, while apHera is an excellent 300 Å polymer-based particle. These two phases are both ideal to separate large and small molecules, which is necessary for complex biological samples (Figure 1). Ascentis® and Discovery® columns are available with small inner diameters for micro-flow applications.

Column performance of Discovery BIO Wide Pore (upper) and Astec apHera C18 (lower) toward 4 small test molecules compared to peptide mixture described in Figure 2.

Figure 1.Column performance of Discovery BIO Wide Pore (upper) and Astec apHera C18 (lower) toward 4 small test molecules compared to peptide mixture described in Figure 2.

Important qualities of mobile phase additives

We recently reviewed the subject of mobile phase additives for LCMS in a 5-part series in Analytix1-5. Stated simply, the challenge of using ionic mobile phase additives is to balance their positive effects on the chromatography against their detrimental effects on the MS detection. Ionic mobile phase additives like trifl uoroacetic acid (TFA) control the pH control complex with oppositely charged ionic groups to enhance reversed-phase retention, or suppress unwanted interactions. However, typical concentrations of TFA (0.1% v/v) have high surface tension and prevent efficient spray formation. Also, TFA ions in the gas phase ion-pair with the peptide’s basic groups suppressing their ionisation and reducing sensitivity.

An easy way to minimise suppression effects is realisable with solvent blends containing 0.1% (v/v) formic acid and 0.01% (v/v) TFA. They both offer good separation conditions and high MS sensitivity and are ideal partners for Discovery® BIO Wide Pore and Astec apHera™ columns.

Alternatives to TFA for LC-MS of proteins and peptides include formic and acetic acid (for low pH), ammonium acetate (for neutral pH) and ammonium bicarbonate (for basic pH). We offer a wide choice of high-purity additives, solvents and convenient additive-solvent blends. Their purity, especially the absence of sodium ions, ensures clean baselines and the absence of adducts that complicate the mass spectra (Figure 2).

Effect of sodium adduct formation on MS spectral quality

Figure 2.Effect of sodium adduct formation on MS spectral quality Detected (upper) and calculated (lower) mass spectrum of human ACTH fragment 18-39 m/z ([M+3H]3+)=822.5 Da. The upper mass spectrum shows a very low abundance of sodium adduct [M+2H+Na]3+ with a m/z ratio of 829.8 Da, demonstrating the purity of the LC-MS solvents.

Mass calibration and performance test standards

In addition to columns and solvents, mass spectroscopy/spectrometry requires solutions of well-defi ned compounds to calibrate the m/z ratio. Molecular weight is often used to aid in protein identifi cation. High-purity calibrants must be used to obtain mass precision in the ppm range, whether these are protein/peptide standards for molecular weight calibration, or small molecules like caffeine, reserpine or uracil, for RP column performance tests. We have designed and developed standards and test mixtures for HPLC, LC-MS and MALDI to calibrate, maintain and troubleshoot the corresponding instruments. Together, they are powerful tools to calibrate the entire LC-MS system and test the performance and suitability of the HPLC column in the same step.


Emmert J. 2006. Mobile Phase Additives for LC-MS, Part 1: Acids – The Most Common Choice. Analytix.(2):8-9.
Emmert J, Rück A. 2006. Mobile Phase Additives for LC-MS, Part 2: Techniques to Overcome the Ionization Suppression Effects of TFA. Analytix.(3):16-17.
Emmert J, Leitner A. 2006. Mobile Phase Additives for LC-MS, Part 3: The Neutral Salts. Analytix.(4):9-11.
Emmert J, Wälti T. 2006. Mobile Phase Additives for LC-MS, Part 4: Special Case - Sodium Adduct Formation. Analytix.(5):6-7.
Emmert J, Köhling R. 2007. Mobile Phase Additives for LC-MS, Part 5: The Bases – Reverse Buffering, Negative and Reverse Ionization. Analytix.(6):4-6.
Sign In To Continue

To continue reading please sign in or create an account.

Don't Have An Account?