Gradient-elution parameters in capillary liquid chromatography for high-speed separations of peptides and intact proteins

Axel Vaast, Eva Tyteca, Gert Desmet, Peter J. Schoenmakers, Sebastiaan Eeltink

INTRODUCTIONGradient-elution

•an important technique for the separation of complex mixtures of analytes with large molecular weights

• the mobile phase composition is changed during the separation process

•useful for impurity profiling, degradation studies, as well as analysis of complex herbal extracts where analytes are having mix polarities

INTRODUCTIONParameters

•Elution

- LSS

- NLSS

•Gradient slope (β)

•Mobile-phase composition - solvent strength

•Column (Stationary)

•Retention factor at the Moment of Elution (ke)

INTRODUCTIONLiquid Chromatography-separation occurs based on the interactions of the sample with the mobile and stationary phases

Capillary Liquid Chromatography-pumps materials at high pressure through a separation column to identify, quantify, and purify compounds of interest

- a smaller separation column (<0.3mm)

Materials and MethodsChemical and Reagents - Acetonitrile (CAN, HPLC supra-gradient quality) : p

- Acetone (≥99.5%) :p and np

- Formic acid :p

- Uracil (99%)

- Deionized water

- Lyphophilized 5 peptide sample

Materials and MethodsInstrumentation and HPLC Conditions

Mobile –phase composition (isocratic mode) - 75:25% (v/v) ACN:H2O w/ 10nL injection of uracil

Column Dead time

t0,monolith = 0.272 min

t0, fused core = 0.272 min at 4μL/min

Materials and MethodsLinear ACN – mobile phase

A : 0.05% (v/v) aq FA

B: 80% (v/v) aq ACN w/ 0.04% (v/v) FA

Dwell Volume

B: 80% (v/v) aq ACN w/ 0.04% (v/v) FA spiked w/ 0.3% acetone

Dwell Time0.282 min (at 4μL/min)

Materials and MethodsGradient experiments conducted

Gradient composition:

Φ0 = 0.008 ACN Φf = 0.640 ACN

Samples were prepared

lypophilized 5 peptide sample dissolved in 1 mL of 1% B

conc. range of 1-2 μg/L for each cpd

intact proteins were injected w/ an individual conc. of 2.5 ng/ μL

Materials and MethodsSoftware Data Analysis

- solver-routine in Microsoft Office Excel 2007for the retention relationship

Results and Discussion Determination of elution parameters: LINEAR MODEL

- Reverse phase Chromatography : linear relationship

ln k = ln kw − S Φ

This model is characterized by two elution parameters; kw, which represents the extrapolated value of k for Φ= 0 (i.e. pure water) and S, the solvent-strength parameter describing the slope of the linear relationship between k and Φ (constant for a given compound and organic solvent).

Results and Discussion Determination of elution parameters: LINEAR MODEL

Results and Discussion Determination of elution parameters: NON LINEAR MODEL

When comparing the results obtained from the linear model and from the Neue–Kuss model (NLSS) , a more accurate fit was obtained in the latter case.

Results and Discussion Comparison of MODELS

- the non-linear model allowed better predictions in all cases

- When extending the prediction range to separations with a very steep slope, of both peptides and proteins with a respective optimized gradient span, the non-linear model stood out as the more accurate one

- the propensity of non-linear models for predicting high speed separation conditions with more accuracy

ConclusionPurpose of study: Two-fold

(1) the dependence of the retention behaviour of such large compounds (peptides and proteins) in terms of the mobile-phase composition was investigated using a conventional linear model (LSS) and the Neue–Kuss model

ConclusionPurpose of study: Two-fold

(2) Even though the LSS-model is shown to allow accurate retention-time predictions under conditions encompassed by the initial runs, only the Neue–Kuss model allowed the prediction of high-speed separations with a high degree of confidence (<2%)

Conclusion- LSS-model lead to the conclusion of peak broadening

rather than peak sharpening

- the use of the more accurate non-linear model showed the occurrence of a clear sharpening effect

This effect predominantly occurs with high-speed separations. No significant effects of system broadening, temperature, and the stationary-phase (pore) structure on compression could be identified