Polystyrene-divinylbenzene (PS-DVB) trap columns have been evaluated for microcolumn switching applications. In contrast to traditional stationary phases, which consist of packed particles, the monolithic separation medium is made of a continuous, rigid polymeric rod with a porous structure. The absence of intraparticular void volume increases separation efficiency, allowing for faster separations. Column lifetime is higher compared to packed columns.

 Figure 1: Direct injection (b) vs microcolumn switching with monolithic trap column of a cytochrome c digest (a) with 1 pmol injected.			

This article demonstrates that the use of monolithic trap columns for preconcentration and desalting of peptides and proteins does not negatively influence chromatographic performance or sample recovery. Sample capacity of the monolithic trap columns (200µm i.d. × 5 mm) is 100 pmol for both peptides and proteins. 

Figure 2: Separation of 16 intact proteins (5 ng each injected) on a monolithic capillary column after preconcentration on a monolithic trap column.			

LC system: UltiMate Plus nano LC system, Switchos 

Trap column: Monolithic trap column, PS-DVB, 

Loading solvent: Water with 0.05% hepta fluorobutyric acid 

Analytical column: Monolithic capillary column, 

  (b) Water, acetonitrile (50:50%, v/v), 

Gradient:  0–70% B in 7 min for peptides

Direct Injection vs Microcolumn Switching

The influence of the monolithic trap column on chromatographic performance was evaluated by the separation of tryptic peptides of cytochrome c. Figure 1 shows a comparison between direct injection and microcolumn switching with a monolithic trap column. Table 1 lists peak widths at half height (PWHH) for the tryptic peptides with and without preconcentration. 

Table 1: PWHH for Tryptic Peptides of Cytochrome c Separated on Monolithic Columns			

Protein Separation After Preconcentration Using Monolithic Columns

Figure 2 shows the separation of 16 standard proteins on a monolithic capillary column with sample loading on a monolithic trap column. PWHH were typically between 4 and 8 s. 

The experiments demonstrate that monolithic trap columns can be used for preconcentration and desalting of samples consisting of peptides and proteins, without negatively influencing the chromatographic performance or recovery of the compounds. The sample capacity of the monolithic trap columns (200µm i.d. × 5Xmm) is in the range of 100 pmol for both peptides and proteins (data not shown). 

Articles

Medical/Biological

PS-DVB Monolithic Columns Applied in A Microcolumn Switching Set-up for the Analysis of Peptides and Proteins

Dionex has developed an autosampler with very precise sample preconcentration capabilities. When analyte detection is no longer possible even with a large loop injection, sample preconcentration (loading a sample through a concentrator column and eluting the concentrate) is used. Although sample preconcentration is not new, the challenge has been to match an appropriate concentrator pump with the accuracy requirement of the application and fully automate the process. As detection limits are reduced, non-reproducibility of the pumping mechanism can lead to significant error and a high relative standard deviation for the analyte of interest. The Dionex AS uniquely pairs a very highly precise syringe pump with the capabilities of an autosampler to allow sample preconcentration in an automated fashion for up to 49 samples.  

The "concentrate" feature on the new AS allows the user to concentrate up to 8 mL of sample with full automation capability. The actual "concentrate" step uses the same mechanism that the autosampler uses to make traditional sample loop injections, albeit with a considerably higher volume than loop loading. With "concentrate", sample is loaded onto a concentrator column (up to 150 psi backpressure) and RSDs of below 0.5% are routinely achieved. The precision is related to the volume delivered, flow-rate across the concentrator and the capacity of the concentrator. The AS syringe pump is able to deliver precise, repeatable volumes onto the concentrator despite possible changes in backpressure. Figure 1 illustrates the use of the AS in determining low concentrations of perchlorate (5 ppb) via sample preconcentration. In this example, 5 mL of sample were injected over a concentrator column (TAC-ULP) to achieve the desired detection limits. RSDs for perchlorate at this concentration are less than 1%.  

 Figure 1: Using the AS to determine low concentrations of perchlorate via sample preconcentration.			

With the Dionex AS, chromatographers now have automated sample preconcentration without the need to manually feed sample into a pump. This translates to direct labour savings as well as improved analysis reliability.  

General

Dionex has developed an autosampler with very precise sample preconcentration capabilities. When analyte detection is no longer possible even with a large loop injection, sample preconcentration (loading a sample through a concentrator column and eluting the concentrate) is used.

The Application Notebook-12-01-2005

PS-DVB Monolithic Columns Applied in A Microcolumn Switching Set-up for the Analysis of Peptides and Proteins

Precise Sample Preconcentration Coupled with Automated Sampling