Cast vs. Skived PTFE Headspace Septa

In headspace analysis, it is of utmost importance that a tight seal is formed between the septum of the cap and the vial. If there are even small gaps present in the seal, some of the analyte will escape through these gaps and lead to low recovery. In order to avoid problems of this kind, the quality of the septa should always be considered.

                There are two manufacturing methods for PTFE used in headspace caps. The first method is skiving. In this technique, a long tube-shaped portion of PTFE material is mechanically cut at regular intervals to produce the circular septa discs used in the final headspace cap product. The second method is known as casting. Here the PTFE material is poured into molds in the shape of the headspace septa. Skiving is the cheaper of the two techniques because quality molds are expensive to manufacture. However, the skiving process is known to produce widespread defects in the material surface. On the edges of the material where the PTFE contacts the vial when sealed, these defects cause gaps in which volatile compounds may escape.

                The difference in quality between these two septa types is readily apparent under magnification. Figure A below shows the PTFE side of a cast septum used in MicroSolv HeadSpace Crimp Caps (catalog # 95025-07-1S). The surface here has only a few minor imperfections. In Figure B, the PTFE surface of a market leading skived headspace cap is shown. Here the surface has significant grooves all throughout the material. These grooves are the result of skiving techniques.

 For more information on MicroSolv HeadSpace Crimp Caps, click here.

Figure A

 

Figure B

 

Aqueous Normal Phase: Not Just for Hydrophilic Compounds

                In HPLC analyses of hydrophobic molecules, reversed phase is the most commonly used separation method. For hydrophilic analytes on the other hand, Hydrophilic Interaction Liquid Chromatography (HILIC) is used in some cases. However, the drawback to these two retention modes is that they cannot simultaneously retain both hydrophilic and hydrophobic analytes in the same run.

                This is in contrast with Cogent TYPE-C Silica™ columns, in which can retain both types of compounds with the same column. In addition to conventional reversed phase, the columns can retain by Aqueous Normal Phase (ANP) as well. What may be surprising is that even hydrophobic analytes can be retained by an ANP mechanism. Cyclobenzaprine for example has a reported log P of approximately 4.6–4.9 and yet still shows excellent ANP retention, as shown in the chromatogram below. This behavior has been observed for hydrophobic peptides as well and has been published in a 2011 article in the Journal of Chromatography A.

                What this means for the practicing chromatographer is that in some cases it is possible to retain both types of compounds not just with the same column but in the same run. This feature is very useful for any analysis that involves compounds encompassing a wide range of polarity, such as metabolomics. The work of two columns could be done in this case with only one.

How Choice of Autosampler Vials Can Affect Quantitation

                The modern analytical laboratory makes use of more sophisticated analytical instrumentation than ever before, leading to lower detection limits and hence a need for more accurate quantitation.  One aspect of the analytical process that is often overlooked is the importance of the autosampler vial. Basic solutes are known to electrostatically adsorb with surface silanols on conventional glass and therefore affect quantitation. This effect becomes significant at lower concentrations.

                So while instrumental detection capabilities have greatly improved in recent years, autosampler glass technology has often not kept pace in many cases. However, unique glass chemistry is available from MicroSolv which is referred to as Reduced Surface Activity (RSA™) Glass. This material is virtually free of surface silanols and therefore better preserves the quantitative characteristics of the analytes.

                A study compared the peak areas of the same 5ppm thiamine standard solution in two different autosampler vials. One was made of conventional glass sold by a leading supplier and the other was MicroSolv RSA™ glass. The solutions were injected immediately after transferring to the vials and again after four hours. The conventional glass showed a 32.6% loss in peak area compared to only 3.1% in RSA glass (see below peak overlays). Thiamine was selected due to the cationic nature of the compound, which would have a high affinity for electrostatic interaction with any silanols on the glass surface. Compounds without basic functional groups did not show loss in peak area with either vial.

RSA Glass Vial Product Page

 

How Analyte Loading Affects Peak Shape & Method Development

Different modes of chromatography require different thinking. In reversed phase (RP), the amount of analyte you can load onto the column without affecting retention or peak shape is not necessarily the same as with Aqueous Normal Phase (ANP) mode. Analytes in reversed phase solvents have different solubilities than in ANP and the retention mechanisms between the two are different as well.

                A given vitamin analyte for example may be retained in both RP and ANP, but the concentration and/or injection volume limits of each mode should be considered separately. An injection volume of 20 µL may be fine for a particular RP method but the same injection volume could produce a tailing peak in ANP under certain conditions. The concentration should also be considered, since the key aspect is the total amount of analyte in a given injection plug. An analyst that observes the tailing peak may incorrectly attribute it to silanol interactions and spend unnecessary amounts of time investigating other mobile phase conditions to reduce the tailing. In this case, understanding the different load limits of the column can make method development faster and easier by anticipating problems before they are observed.

                A study was done on a water soluble vitamin in ANP mode in which a series of runs was performed and only the injection volume was varied with each method. Under these conditions, it was observed that while an injection volume of 1 µL produced a symmetrical sharp peak, progressively increased tailing and decreased retention were observed with higher injection volumes. The 10 µL peak was significantly lower in retention and produced unacceptable tailing.

 

http://kb.mtc-usa.com/article/AA-00876

Analyzing Morphine by HPLC Can Be Done Easily

Separating compounds of special interest like morphine can be difficult but also fun. I find it very challenging because amines such as morphine have a tendency to interact with residual silanol groups in an HPLC column, which shows up in the chromatogram as a tailing analyte peak.

Accurate determination of peak area assumes a symmetrical Gaussian peak shape and therefore tailing comprises the reliability of the data. Peak tailing can also overlap into neighboring peaks as well, creating other problems. The USP assay methods for morphine require ion pair reagents to prevent the analyte–silanol interactions. However, these reagents cannot be used in LC-MS and limit the applications of the assay methods.

We presented a method using our innovative Diamond Hydride™ column that not only is completely LC-MS compatible, but also produces excellent retention, efficiency, and peak shape for this problematic analyte. This has made life as a chromatographer easier for me when my boss gives me such a challenge.

http://kb.mtc-usa.com/article/AA-01128