Improving the performance of Micro LC columns

Micro LC columns offer chromatographers the opportunity to improve sensitivity or use lower flow rates due to their smaller internal diameter. The downside to reduced column diameter, is in order to maximize efficiency, sources of band broadening need to be minimized and sample preparation is critical to improve column lifetime.

Sample Preparation

• Using a trap and elute format will provide cleaner samples for injection on to the analytical column and improve column lifetime
• Utilizing sample preparation techniques such as solid phase extraction (Strata-X^® SPE products) or accessories (Phenex™ Syringe Filters) to minimize the injection of unwanted contaminants onto your system and column can also prolong column lifetime

Trap Configuration

Phase selectivity and the direction of mobile phase flow onto and off the trap column, will all contribute to column efficiency and lifetime. The choice of an appropriate stationary phase for your trap will help to improve separation, whereas direction of flow during your trap and elute method can improve column lifetime by minimizing particulate build up when used in the forward direction.

Forward Elute

In the forward direction, your injection loads the sample onto the trap in the forward direction. While this is still pumping to waste you can here carry out a washing step if you wish to. Once you alter the trap valve the sample is pumped from the trap column onto the analytical column. With forward elution the sample has to pass all the way through the trap column to reach the analytical column and the stationary phase in this trap column is more impactful on separation than in the reverse elute mode. It also means any insoluble matter stays trapped on this column and your Micro LC column is much better protected.

Reverse Flow

With reverse elute the sample is loaded and washed in the same way, but now when we switch the valve the gradient is pumped in the reverse direction, eluting the sample back off the trap in the same direction it was loaded onto it. This makes the packing material less important in terms of overall selectivity as the flow path is shorter, but it also means insoluble matter has the potential to be flushed back out of the trap and into your Micro LC column. In the reverse direction you can also benefit from using a wider ID trap; which can be loaded at a faster flow rate; accelerating this step if you have a time sensitive application.

What’s in a Name? – Decipher the Code of Dibenzodioxins and Dibenzofurans

“I am trying to separate my 1238 from my 2378!”
We can make sense of this by first numbering the positions around our dibenzo compounds to which chlorines may bond. Notice that the tertiary carbons are not numbered. Be careful as to when you are discussing polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzodioxins (PCDF). The next consideration is the prefix that denotes the number of chlorines that are bonded to the dibenzo compound. Environmental methods are typically concerned with PCDD and PCDF compounds with four or more chlorine substituents, for which the prefixes and abbreviations are shown in Table 1. The “octa-chlorinated” analogues do not require numbering, as all of the substituent positions are bonded with a chlorine substituent.

Table 1 – Examples of PCDDs and PCDFs

# of Cl Substituents	Examples
(4) Tetra – T	Example: dioxin (PCDD) Name: 2,3,7,8-TCDD
(5) Penta – Pe	Example: dioxin (PCDD) Name: 1,2,3,7,8-PeCDD
(6) Hexa – Hx	Example: furan (PCDF) Name: 1,2,3,4,6,8-HxCDF
(7) Hepta – Hp	Example: dioxin (PCDD) Name: 1,2,3,4,6,7,8-HpCDD
(8) Octa – O	Example: furan (PCDF) Name: OCDF

PCDD and PCDF compounds are often referred to by the nomenclature demonstrated in Table 1. There are occasions during routine testing when these compounds are referred to simply by the position of the chlorine substituents, such as “separate a 1238 from a 2378.” Clarify that the context specifically pertains to either PCDD compounds or PCDF compounds, especially when speaking with members of other laboratories or organizations.

Thank you to the analytical chemists and laboratory technicians who monitor environmental samples for PCDD and PCDF compounds, along with the related herbicide and PCB compounds. Hopefully we have dispelled the ambiguity that may surround “dioxin and furan” analysis for any newcomers to the topic.

Why are my recoveries greater than 100%?

When performing analyte extraction with SPE we expect 100% recovery. If recovery is lower this would suggest there is an issue with retrieval of the analyte from the sorbent but what are some of the reasons you may see a recovery greater than what you were expecting (or over 100%)

• Co-eluting Interferences
• Contaminants from the solvent or sorbents selected
• Internal standard is compromised making your reference point inaccurate
• Recovery calculations are inaccurate

Co-eluting interferences: These are typically present in the matrix and the goal of the SPE step is to remove them. If you are experiencing recoveries greater than 100% the first step would be to check the eluent using an orthogonal analytical technique to identify that the increase recovery is a result of a co-elution. Where a co-elution is identified there are a few possible solutions.

1. Modify the wash steps of the SPE method to selectively remove the impurity during washing
2. Select an alternative SPE sorbent which will better remove these impurities. This is easier if the analyte / impurity can be selectively ionised as an ion-exchange sorbent will very often afford cleaner extracts than either reversed or normal phase solutions
3. Adjust your analytical method to resolve the 2 peaks analytically negating the necessity to remove this impurity during the SPE step.
4. Run a blank plasma sample, this will allow you to ascertain the impurities present and use this blank in the calculations to subtract from your recovery

Contaminants from the solvent or sorbent: These will typically be impurities present in the starting materials which are similar in nature to your compound of interest and as a result co-elute with it during SPE and subsequent analytical analysis. You can follow the steps above to remove co-eluting impurities or alternatively as this is not a impurity present in your matrix you can identify the level present in the sorbent using blank runs and subtract this from your main peak during the calculation. Prior to addition of your sample if you equilibrate your sorbent with the elution solvent this should flush out potential contaminants from the sorbent prior to adding the sample.
For impurities introduced from the solvent, typically switching the solvent should resolve this.

Internal standard is compromised: If your internal standard is not showing 100% recovery your subsequent calculations will be inaccurate. It is always good practise to use an internal standard similar in nature to your compound of interest as this will give the more consistent reference point for your method. If there is concern your internal standard is not showing good recovery from the SPE material using your method you can use an external standard which is added to the elution solvent to calculate absolute recovery.

Recovery calculations are inaccurate: It is important to follow this step wise to ascertain where the errors are appearing.

1. Calculation Errors- Check all calculations and account for all dilution factors
2. Integration Errors- Reintegrate all peaks and use manual calculations to confirm
3. Analytical method errors – Ensure the analytical method used allows correct integration and calculation. Ensure no strong solvents are used for injection and run a calibration across a number of dilutions to ensure linearity. Troubleshoot your analytical method to determine any factors which may lead to poor reproducibility
4. Ensure detections selected are appropriate and working correctly.