How is the amber color produced in the polypropylene/glass amber autosampler vials?

The amber color present in some of the autosampler vials is created by organic or inorganic pigments or dyes. In glass vials, the amber color is from iron oxide, added in less than 1% concentration.
       In plastic (polypropylene) vials, the color is produced from a proprietary organic compound present in mostly less than 0.1% concentration so no iron is used making it appropriate for Ion Chromatography. At concentrations greater than ~2%, the vial becomes completely opaque. The full list of items which have an amber color is given as follows:

Catalog Number	Material
9502S-PP-A	plastic
97040-0AV	glass
9502S-WAV	glass
97060-0AV	glass
95002-0AP-A	glass + PP
95010-0AV	glass
95020-0AV	glass
95025-CT-10A	glass
95025-CT-20A	glass
95025-PE-20A	glass
9502C-0-WAV	glass
9502C-WAV	glass
9503S-WAV	glass
9504S-0AV	glass
9504S-WAV	glass
9532C-TS-A	TPX
9532S-TS-A	TPX
97015-AV-08	glass
97015-AV-12	glass
97018-SP	PP + Butyl/PTFE
97020-0AV	glass
97030-0AV	glass

     The purpose of the amber color is for cases in which light sensitive compounds are involved. Folic acid for example is sensitive to photo-oxidation and therefore the effects of ambient light could be detrimental to accurate analyte quantitation.
     There are two types of chemicals which are introduced to a substrate to produce color: pigments and dyes. Although the two terms are often used interchangeably in everyday language, they are actually distinct. Pigments are insoluble in the substrate in which they are infused and are present as a dispersed suspension in the material. Dyes on the other hand are soluble in the substrate and are present as a solution. In the amber products discussed above, the iron oxide in glass is a pigment and the proprietary organic compound in polypropylene is a dye.
      A wide variety of colors besides amber can be produced in glass depending on the colorizing agent used. The green color used in wine bottles (Fig. 1) for example is produced by a combination of iron oxide and chromium (in the form of chromic oxide or potassium dichromate). Copper produces a turquoise color notable for use in Egyptian Blue, a widely used synthetic pigment in antiquity (Fig. 2). Cobalt gives a deep blue color which has been found in many examples of ancient Chinese porcelain (Fig. 3). Also, glass coloring is not necessarily produced by the addition of pigments or dyes. Due to the Tyndall effect for example, color in glass can be achieved by light scattering in a suitable medium.

Fig. 1. Coloration in wine bottle

 

Fig. 2. Example of Egyptian Blue in an ancient artifact

 

Fig. 3. Cobalt used in Chinese porcelain.

Clear Polypropylene 2ml Vial Information
Amber Polypropylene 2ml Vial Information

More Differences Discovered between Silica-C™ and Ordinary Silica

The Cogent TYPE-C™ line of HPLC columns are all based on a unique type of silica in which surface populated with  silica hydride groups (>95%). The difference has a significant impact on a variety of chromatographic aspects with many benefits for the users. In order to demonstrate these differences, we compared a plain silica column with an underivatized Silica-C™ column. The method conditions are shown in Table 1. A simple isocratic method with two test solutes was chosen so that meaningful comparisons could be drawn regarding efficiency and so on.

                The chromatograms obtained are shown in Figure 1 and the data is presented in Table 2. What is most notable is that the silica column could not distinguish amongst the two test solutes under these conditions, while baseline resolution is obtained using the Silica-C column. This illustrates how the hydride surface can dramatically change the behavior of the column. In terms of peak shape, the silica column exhibits significantly more tailing, possibly due to the influence of silanophilic interactions with the amine groups of the test solutes. Finally, efficiency on the Silica-C phase surpasses the silica material as well for chromatographers.

 

Table 1. Method Settings

Parameter	Description
20% Solvent A	DI H2O + 0.1% formic acid
80% Solvent B	Acetonitrile + 0.1% formic acid
Flow Rate	1.0 mL/min
Injection Volume	0.5 µL
Detection	UV 210 nm
Sample	Phenylglycine and phenylalanine, 10ppm each

 

Table 2. Obtained and calculated chromatographic values

	HPS SILICA		SILICA-C
	Phenylglycine	Phenylalanine	Phenylglycine	Phenylalanine
tR (min)	4.504	4.504	7.276	8.484
selectivity	1.00		1.22
Rs	0.00		3.60
Tf	2.37	2.37	1.11	1.29
N/column	1967	1967	9097	8645
N/meter	13113	13113	60647	57633

 

Figure 1. Overlay comparison of column data. Peak 1) phenylglycine, peak 2) phenylalanine.

 

 

 

Using temperature changes for separations of terpenoids with the Cogent Bidentate C18 and UDC Cholesterol HPLC columns

Temperature is a useful variable to consider in HPLC separations. Control of temperature can reduce run times, alter selectivity, and change analyte efficiency. In this study of three terpenoids (bexarotene, tretinoin, and tazarotene), we compared the Bidentate C18™ and UDC-Cholesterol™ columns at different temperatures in an attempt to gain insight into their retention behavior. The method conditions are shown in Table 1. Figures 1 and 2 show the retention times obtained for the three analytes as a function of temperature using each column. The “UDC” column is known to exhibit shape selectivity for some analytes, and we can see that the elution order is different when compared to the Bidentate C18.

                The Bidentate C18 shows greater selectivity between the critical peak pair at higher temperature whereas the UDC shows the opposite trend. Since shape selectivity effects are known to be more pronounced when the UDC stationary phase moiety is more rigid, it makes sense that the lower temperatures would produce greater separation between the analytes. The Bidentate C18 stationary phase on the other hand does not have this property and therefore the effect is not the same.  In fact, when using the Bidentate C18 at 15 °C where the UDC had shown the best separation, bexarotene appeared as a shoulder peak on the tazarotene peak.

                Peak efficiency initially increased with increasing temperature until it reached a maximum and then decreased (see Figure 3). The drop-off was more significant on the UDC column, suggesting that rigidity of the UDC moiety may play a more important role in the retention than the Bidentate C18. The maximum values were obtained in the range of 35–40 °C.

                Knowing how temperature affects the separation can lead to more informed choices during method development. Resolution depends on both efficiency and selectivity, but their optimum values are not always the same; the selectivity on the UDC was best at 15 °C but the efficiency was highest at 35 °C. The analyst should consider the relative importance of each effect that the temperature has on the chromatographic data when choosing an optimum column temperature.

Table 1. Method conditions for the separations.

Parameter	Details
Solvent A	DI water + 0.1% formic acid
Solvent B	Acetonitrile + 0.1% formic acid
Flow Rate	1.0 mL/min
Gradient	0–1 min hold at 30%B, 1–24min to 100%B, 24–25min to 30%B
Detection	UV 254nm
Injection Volume	10µL

 

Figure 1. Retention as a function of temperature on the UDC column.

 

Figure 2. Retention as a function of temperature on the Bidentate C18 column.

 

Figure 3. Efficiency of tazarotene as a function of temperature on both columns.

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.