Use the “Subscribe” link at right. It just takes a few seconds!
Comparison between Different Stationary Phases for the Separation of Phthalates using Gas Chromatography-Mass Spectrometry (GC-MS)
Dan Li, Rebecca Stevens, and Chris English
Phthalates are widely used as plasticizers in a variety of industry products. However, some phthalates are considered as endocrine disruptors associated with a number of problems, including birth defects, high blood pressure in children, pregnancy-induced hypertensive heart diseases, respiratory problems, and obesity. The European Union (EU) and United States Environmental Protection Agency (US EPA) have restricted the use of the most harmful phthalates (see Table 1).
A commonly used technique for phthalate analysis is gas chromatography-mass spectrometry (GC-MS), which is simple, fast and inexpensive. GC-MS also provides additional mass spectral information and thus serves as an ideal instrumental platform for phthalate identification and quantification. Selection of GC columns is critical, because each stationary phase has a unique selectivity, which affects the relative elution order and resolution. Phthalate separation is challenging due to structural similarity. For instance, many phthalates share a common base peak ion (m/z 149) which makes identification of coeluting phthalates difficult. Technical grade mixtures and isomers further complicate identification of target phthalates.
Researchers have evaluated different stationary phases for phthalate analysis. A recently published review summarized the most used GC and liquid chromatography (LC) columns. According to the literature, GC-MS has higher sensitivity compared to LC-MS for phthalate determination, and the most commonly employed GC columns in descending order of popularity are 5-type, XLB-type, 35-type, 17-type, 50-type, and 1-type.
Separation specific to a stationary phase is achieved by adjusting conditions. The Pro EZGC® program is a fast modeling software which can optimize GC parameters (e.g., carrier gas type, flow rate, temperature program, column dimensions, and guard column) to produce the shortest run time on a given type of stationary phase. In this study, libraries of 37 phthalates (see Table 2 for phthalate names) were built into the Pro EZGC® program for seven of the most frequently used column phases: Rtx®-440, Rxi®-XLB, Rxi®-5ms, Rtx®-50, Rxi®-35sil, Rtx®-CLPeticides, and Rtx®-CLPesticides2. These stationary phases were evaluated for the analysis of both regulated and non-regulated phthalates.
Table 2. Elution times for Phthalates on various Restek GC columns.
The standard EPA Method 8061A Phthalates Mixture (cat. #: 33227) consists of 15 components, each at a concentration of 1,000 μg/mL. Benzyl benzoate (cat. #: 31847) was used as the internal standard. All other phthalate standards were purchased from Chem Service (West Chester, PA). The GC-MS analysis was performed on a Shimadzu GC-MS QP2010 Plus instrument. The GC-MS was equipped with one of seven Restek columns using 30 m×0.25 mm×0.25 µm dimensions (0.20 µm for Rtx®-CLP2 column) (see Table 1 for the column types). The GC-MS experimental parameters were listed in Table 3.
Table 3. GC-MS parameters meters
Standards and tested samples were dissolved and diluted in methylene chloride. Standard solutions were prepared at 50 μg/mL (80 μg/mL for benzyl benzoate). During sample preparation, plastics were strictly avoided; all preparation work was performed using glassware (volumetric flask, syringe, vial, etc).
A direct comparison between the columns for the separation of EPA and EU regulated phthalates was performed. The elution times of seven different phases were predicted by the Pro EZGC® program under the same optimized GC conditions (Table 1). Coelutions were counted as compound pairs with a resolution less than 1.5. The total analysis time is less than 6 min under these conditions. In order to confirm the prediction, chromatograms of each stationary phase were collected under the same optimized condition (Figure 1). Because the column length is not exactly 30 meters long as in the simulation, the retention times are slightly different from the prediction. The elution orders and coeluting pairs were exactly the same as predicted. Among the seven phases, Rtx-440, Rxi-XLB and Rtx®-CLP and Rxi®-35 sil provided baseline separation for all EPA and EU listed phthalates. The two isomers of bis(2-ethylhexyl) phthalate were not resolved on the seven phases. The elution order was comparable on the Rtx®-440, Rxi®-XLB, Rtx®-CLP and Rxi®-5ms columns. Interestingly, differences in the elution orders were observed on Rxi®-35 sil and Rtx®-50 phases. Most notably, the elution orders of 4 pairs of phthalates changed on Rxi®-35sil phase, including bis(2-methoxyethyl) phthalate / bis(4-methyl-2pentyl) phthalate (peak 6 and 7/8), bis(2-ethoxyethyl) phthalate / diamyl phthalate (peak 9 and 10), butyl benzyl phthalate / hexyl-2-ethylhexyl phthalate (peak 12 and 13), and bis(2-n-butoxyethyl) phthalate / bis(2-ethylhexyl) phthalate (peak 14 and 15). The Rtx®-440 and Rxi®-35sil columns are ideal as a parallel dual column set for electron capture detector (ECD) analysis, where Rxi®-35sil column serves as a good confirmation column. Rtx®-440 and Rtx®-XLB columns showed the highest resolution in this fast analysis. Peaks that coeluted on other phases were well resolved on Rtx®-440 and Rtx®-XLB columns. For instance pairs that are not resolved on other phases include: butyl octyl phthalate and dicyclohexyl phthalate (peak 15 and 16) on Rxi®-5ms; butyl octyl phthalate and butyl benzyl phthalate (peak 15 and peak 12) on Rtx®-50 column; and di-n-butyl phthalate and bis(2-ethylhexyl) phthalate (peak 6 and peak 7,8) on Rtx®-CLP2 column. It is challenging to separate technical isomer mixtures, such as diisononyl phthalate and diisodecyl phthalate (peak 18 and peak 19). Fortunately, unique extracted ions are available for identification and quantification, i.e., m/z 297 for diisononyl phthalate, and m/z 307 for diisodecyl phthalate (see Figure 1).
A comprehensive comparison between the seven stationary phases for the separation of 37 phthalates (a total number of 40 peaks including 3 isomers) was performed using retention times predicted by the Pro EZGC® program (see Table 2). The GC parameters, specified in Table 3, provided separation of 34 out of 40 peaks on both Rtx®-440 and Rxi®-XLB columns in less than 40 min. The two phases have different coelutions. The chromatogram on the Rtx®-440 column was collected and shown in Figure 2. For some pairs that were not baseline-resolved, the resolution is still adequate for qualitative analysis.
There is no single condition set optimal for all phases. The fast program with the most peaks resolved was selected for the column comparison. Further optimization for each phase can be achieved using the Pro EZGC® program.
The most commonly used seven GC columns were compared for phthalates analysis. The Pro EZGC® program provides flexibility in GC optimization. The superior selectivity and efficiency of Rtx®-440 and Rxi®-XLB columns resulted in a fast runtime in both regulated phthalates and the extended list. With good resolution, higher maximum operating temperature (340 ºC for Rtx®-440 and 360 ºC for Rxi®-XLB), and minimum phase bleed, the Rtx®-440 and Rxi®-XLB columns are the preferred choices for phthalate analysis. A dual column set of Rtx®-440 and Rxi®-35sil is an alternative method for analyte confirmation.
The authors would like to thank Shimadzu Corporation for their consultation with the operation of the QP2010 Plus GC-MS Instrument
 Choi, H.; Kim, J.; Im, Y.; Lee, S.; Kim, Y., The association between some endocrine disruptors and hypospadias in biological samples. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering 2012, 47 (13), 2173-9.
 Nassar, N.; Abeywardana, P.; Barker, A.; Bower, C., Parental occupational exposure to potential endocrine disrupting chemicals and risk of hypospadias in infants. Occupational and environmental medicine 2010, 67 (9), 585-9.
 Trasande, L.; Sathyanarayana, S.; Spanier, A. J.; Trachtman, H.; Attina, T. M.; Urbina, E. M., Urinary phthalates are associated with higher blood pressure in childhood. The Journal of pediatrics 2013, 163 (3), 747-53 e1.
 Werner, E. F.; Braun, J. M.; Yolton, K.; Khoury, J. C.; Lanphear, B. P., The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The HOME Study. Environ. Health 2015, 14, 75.
 Jaakkola, J. J.; Knight, T. L., The role of exposure to phthalates from polyvinyl chloride products in the development of asthma and allergies: a systematic review and meta-analysis. Environ. Health Perspect. 2008, 116 (7), 845-53.
 Hatch, E. E.; Nelson, J. W.; Stahlhut, R. W.; Webster, T. F., Association of endocrine disruptors and obesity: perspectives from epidemiological studies. Int. J. Androl. 2010, 33 (2), 324-32.
 Net, S.; Delmont, A.; Sempere, R.; Paluselli, A.; Ouddane, B., Reliable quantification of phthalates in environmental matrices (air, water, sludge, sediment and soil): a review. Sci. Total Environ. 2015, 515-516, 162-80.
As discussed in previous blogs, the U.S. Environmental Protection Agency (EPA) Method TO-15 is applicable to a subset of 97 volatile organic compounds (VOCs) that are from the list of 189 hazardous air pollutants (HAPs) included in Title III of the Clean Air Act Amendments; however, only a few laboratories are analyzing all 97 components. Most laboratories are evaluating what I refer to as the standard suite of 65 VOCs for TO-15. Some researchers add or subtract a compound or two, but again for the most part everyone is looking at approximately the same 65 analytes. As I indicated last time, NJ made some custom changes and has decided that you need to look at these 10 extra VOCs (on top of the of the aforementioned 65), and that you do not need to look at acrolein and naphthalene. Why… I am not sure, but regardless NJ LL TO-15 specifically calls out 73 VOCs to be evaluated.
So first things first… right? We need to concentrate the aforementioned VOCs and shoot them onto our GC-MS. To accomplish this I utilized the Markes CIA Advantage™ and an Agilent 7890B-5977A GC-MS with the following parameters:
With the aforementioned parameters I injected 250 mL of a 10 ppbv mix and we get the following separation of all 75 VOCs (yep… I included acrolein and naphthalene, because I find them interesting):
As one can see from the above total ion chromatogram (TIC), the 79 VOCs (every run includes 4 internal standards) are separated well in a 22 minute GC run. The only critical coelution found was for chloromethane and n-butane; however, we will discuss the significance of this coelution during the next installment of this blog series, which will cover calibrations. Note: I did not do anything fancy with the GC oven program, as I more or less started off with a SOF and OHR program.
The above TIC does not do justice to how well the Markes CIA Advantage™ and the Rtx®-VMS are trapping, desorbing, and seperating these VOCs, so I have included the following extracted ion chromatogram for ethanol and isopropyl alcohol:
Anyone with some VOC experience can testify to the fact that the above EIC of ethanol and IPA is just plain awesome! So, mission accomplished. We have successfully concentrated and separated the compounds required by NJ (plus acrolein and napthalene, because we can). Stay tuned for next time, when I will talk about calibrating for NJ LL TO-15, which oddly enough… can be tricky because the method stipulates that most of the VOCs have to be calibrate from 0.2 to 40 ppbv.
NJDEP-SRP Low Level TO-15 Series: Part 1 – Let’s talk about ambient air sampling in the Garden State
Considering I was born and raised in Jersey for 28 years, I think this blog series is right up my alley. With that said the following blog series is going to address how a laboratory may meet all of the New Jersey (NJ) Department of Environmental Protection (DEP) Site Remediation Program (SRP) Low Level (LL) USEPA TO- 15 Method requirements with a Markes CIA Advantage™.
See… in typical Jersey fashion the state had to be special [like not using the “new”… which I am guilty of (see above)] and make their own method TO-15, as opposed to just following the prescribed U.S. Environmental Protection Agency (EPA) TO-15, which we have covered in previous blogs. Well, that is partly the truth… the rest of the story is that the NJ LL TO-15 method “provides for a lower reporting limit and additional quality control requirements.” However, customer feedback indicates the method is like traffic on the Garden State Parkway… yeah, you get the idea.
Regardless of opinions, this blog series is going to focus on tackling the differences between the U.S. EPA TO-15 and the NJDEP-SRP LL TO-15. The following is a list, as identified in the method, of what modifications have been made to the U.S. EPA TO-15 in the method NJ LL TO-15:
- Holding times
- Canister types and regulators
- Method detection limits
- Reporting limits
- Clean canister certification levels
- GC/MS tuning and instrument performance check requirements
- GC/MS techniques
- Standard type and concentrations
- Initial and continuing calibration standards
- Laboratory control samples
- Limitation regarding the source of make up air
Some of the aforementioned changes are straight-forward/self-explanatory; therefore, I will not be discussing these changes. Rather, I plan to focus on the issues I have found laboratories to struggle with in an attempt to meet the NJ LL requirements (e.g., calibrating from 0.2 ppbv to 40 ppbv). So… stay tuned to this blog series if NJ LL interests you. And no… Snooki will not be making any appearances in this blog series.
Changing from helium to nitrogen carrier gas in gas chromatography while maintaining separation efficiency and analysis time
In two recent ChromaBLOGraphy posts (see links below) I described the experimental implementation of an idea Jaap de Zeeuw had to use nitrogen carrier gas for GC while maintaining the same separation efficiency and analysis time as what could be achieved by helium carrier gas. Wait, that’s impossible, right?! Not if you go from a 30m x 0.25mm x 0.25µm column using helium to a 20m x 0.15mm x 0.15µm column using nitrogen, as Jaap planned by using our EZGC Method Translator and Flow Calculator. Rather than blather on about it here, I encourage you to read the recent Cover Story article we just published in The Column (October 26, 2015; Volume 11, Issue 19). While there is some overlap with the blog posts for this article, we also included some newly acquired ruggedness data for the 0.15mm x 0.15µm column approach, given that being less tolerant of “dirt” could be one of the downsides of going to a smaller diameter. I think you will be pleasantly surprised at the robustness of our 0.15mm columns!
P.S. If you’ve been reading my last few ChromaBLOGraphy posts, you’ve probably already guessed that the nitrogen 0.15mm column approach benefits by using “shoot-and-dilute” GC.
Dan Li, Rebecca Stevens, Jason Thomas and Chris English
If you don’t have a GC-MS for identification of the EPA regulated phthalates, don’t worry! Here we introduce a parallel dual column set for the analysis of regulated phthalates using µECD. As shown in my previous blog , the Rtx-440 column (cat. # 12923) is a perfect choice for fast phthalate separation and quantification. The Rxi-35sil (cat. # 13823) is a good choice for a confirmation column. The dual column set can separate the 16 target phthalates listed in method EPA 8061A, including the internal standard benzyl benzoate (cat. # 31847), in less than 8 minutes
As seen in the chromatogram below, 4 pairs of phthalate peaks (in red) switched elution orders on the Rxi-35sil column, which provides confirmative information for identification. (Note: the peaks were numbered according to the EPA 8061A method.) Concentrations of the phthalates have been adjusted to get adequate and relatively equivalent response to the µECD detector.
Columns: Rtx-440 30 m, 0.25 mm ID, 0.25 μm (cat. # 12923) and Rxi-35sil 30 m, 0.25 mm ID, 0.25 μm (cat. # 13823) using Rxi guard column 5 m, 0.25 mm ID (cat.# 10029) with deactivated universal “Y” Press-Tight® connector (cat.# 20405-261); Sample: EPA Method 8061A Phthalate Esters Mixture (15 components) (cat.# 33227), hexyl 2-ethylhexyl phthalate, benzyl benzoate (internal standard) (cat. # 31847); Injection: Inj. Vol.: 2 μL split ratio :50:1, Inj. Temp.: 280 °C; Liner: Sky® 4.0 mm ID Cyclo Double Taper Inlet Liner (cat.# 23310.5) Oven: Oven Temp: 150 °C (hold 1 min) to 330 °C at 30 °C/min (hold 2 min); Carrier Gas: He; Detector: μ-ECD @ 330 °C; Notes: Instrument was operated in constant flow mode (3.4 mL/min). This chromatogram was obtained using an Agilent μ-ECD. To obtain comparable results, you will need to employ a μ-ECD in addition to dual columns connected to a 5-meter guard column using a “Y” Press-Tight® connector. Concentrations are as listed.
Compared to the method recommended by EPA 8061A using a 5-type/1701-type column set, the run time using the Rtx-440 / Rxi-35Sil MS columns has been greatly reduced from 40 minutes to 8 minutes. Additionally, the resolution has been greatly improved, especially for bis(4-methyl-2-pentyl) phthalate / bis(2-methoxyethyl) phthalate (peaks 5 and 6) and diamyl phthalate / bis(2-ethoxyehtyl) phthalate (peaks 7 and 8). The Rtx-440 column showed superior selectivity over 5-type columns resulting in a faster runtime.
Overall, we recommend the Rtx-440 / Rxi-35sil dual column set as an ideal solution for fast phthalate separation and identification. Due to flow rate limitations on some mass spectrometers, the short run time on GC-µECD may not be able to be reproduced on the GC-MS.
Figure 1 and Table 1 are from EPA Method 8061A . Column 1 is a 30 m x 0.53 mm ID x 1.5 µm 5-type column. Column 2 is a 30 m x 0.53 mm x 1.0 µm 1701-type column. Temperature program is 150 ºC (0.5 min hold) to 220 C at 5 ºC/min, then to 275 ºC(13 min hold) at 3 ºC/min.
Fast gas chromatographic residue analysis in animal feed using split injection and atmospheric pressure chemical ionization tandem mass spectrometry
In a recent ChromaBLOGraphy post I mentioned that we are doing a campaign on “shoot-and-dilute GC”, also known as split injection GC, at RAFA 2015. A paper just published by Tienstra, Portolés, Hernández, and Mol used split injection with APGC for pesticide and other residue analysis in animal feed to achieve many of the benefits we’ve been extolling for split injection with sensitive detectors. Highlights from their paper were listed as:
Fast GC run time: < 10 min. (JC note: fast cycle time too because of higher initial oven start temp)
Less matrix on the GC column and in the MS source. (JC note: increased uptime)
Better compatibility with acetonitrile extracts (QuEChERS). (JC note: most of solvent is split off before GC column)
Applicable to complex feed matrices. (JC note: APGC with MS/MS is very sensitive and selective)
The 7th International Symposium on Recent Advances in Food Analysis, better known in short as RAFA, starts this week in Prague, The Czech Republic. This is my tenth anniversary for this biennial meeting, where the organizer, Jana Hajšlová, invited me to give the lecture, Using the QuEChERS Sample Preparation Method and GCxGC-TOFMS to Determine Pesticides in Baby Foods, back in 2005 at the 2nd RAFA. One of the main points of that 2005 talk was exploring the use of analyte protectants to quantitatively improve gas chromatography of active pesticides such as omethoate, demeton-S-methyl-sulfone, etc. Example analyte protectants include volatile sugars that are rich in hydroxy functionalities so that when co-injected at high added concentrations with extracts tie up Si-OH and other active spots in the GC inlet liner and the GC column. Not surprisingly given the previous literature reports and my discussions with Steve Lehotay at USDA, I found that at low pg levels some active pesticides in the presence of analyte protectants showed almost a 100-fold response factor increase versus solvent-only standards. Needless to say, when the Maximum Residue Levels for some pesticides in baby food are below 10 ppb, this increase in detectability can be extremely important.
The desire for good instrument sensitivity to determine low pesticide and pollutant residue levels in food and the environment has continued for the last 10 years, and the instrument vendors have made large advances in detecting lower levels, so much so that LC-MS/MS practitioners are expanding their employment of “dilute-and-shoot” methods as a way to avoid ion suppression and other “detector” associated phenomena that lead to sub-par results. While the detectability strides for GC-MS/MS haven’t been as significant, the GC instruments are still getting better (including introduction of APGC and GC-Orbitrap), leading to the possibility of using split injection to relieve some of the GC inlet activity (e.g., omethoate sorption) and reactivity (e.g., captan breakdown) issues faced by residue chemists. We call split injection, “shoot-and-dilute GC”, and are focusing our presentation efforts at RAFA 2015 on this technique, including two where we compare and contrast split injection and the use of analyte protectants. If you can’t join us for the following “shoot-and-dilute” presentations, please request them from either Julie Kowalski (email@example.com) or Jack Cochran (firstname.lastname@example.org).
Julie Kowalski and Jack Cochran; Shoot-and-Dilute Gas Chromatography-Mass Spectrometry: Polycyclic Aromatic Hydrocarbons Quantification in Tea Using Modified QuEChERS Extraction and No Sample Cleanup
Julie Kowalski and Jack Cochran; Shoot-and-Dilute GC-MS/MS: Use of Split Injection for Pesticide Residue Screening to Prolong GC Inlet Liner and Column Performance
Jack Cochran and Julie Kowalski; Shoot-and-Dilute GC-ECD for Analysis of Problematic Pesticides (including Captan and Folpet)
Jack Cochran, Michelle Misselwitz, and Julie Kowalski; Prolonging GC-MS/MS Performance for Pesticide Analysis: Shoot-and-Dilute Injection and Analyte Protectants (Introduction)
Jack Cochran, Michelle Misselwitz, and Julie Kowalski; Prolonging GC-MS/MS Performance for Pesticide Analysis: Shoot-and-Dilute Injection and Analyte Protectants (Ruggedness for Real World Samples)
I mentioned in my last ChromaBLOGraphy post, Phthalate-Free Personal Care Products?, that we used GCxGC-TOFMS to determine phthalates in a Las Vegas Wash water sample. While it doesn’t dive into specific compound identification, we recently published the first demonstration of a near theoretical maximum peak capacity gain for GCxGC (approximately 9x) in an open access Journal of Chromatography A article using that Las Vegas Wash sample extract. You can download the paper for free (click on Download PDF after clicking on the link above). An excerpt from our article is shown below to indicate the significance of the peak capacity increase.
“To put the potential peak capacity gain of GCxGC in perspective relative to 1D GC, consider the following. Peak capacity of an open tubular capillary column is proportional to L/dc where dc and L are the column internal diameter (i.d.) and length. A range of typical low and high efficiency columns in lab GCs might span from a 15 m × 0.32 mm to a 40 m × 0.10 mm capillary column. The relative difference in peak capacities between these two columns is approximately 3. Compare this entire range of 1D peak capacities with the fact that GCxGC has the potential to achieve a 10-fold or larger peak capacity gain in the same time frame. For comparison, what would it take in terms of analysis time and resources to obtain a 10-fold peak capacity gain by simply increasing the length of a 40 m × 0.10 mm column in 1D GC analysis? It would require a 100-times longer column (4 km instead of 40 m), 10-times higher inlet pressure (about 100 atm instead of about 10 atm for helium), and the analysis time would be 1000 times longer (1.5 months instead of 1 h or so for helium). Even an incremental 2-fold peak capacity increase would be beyond currently available 1D resources (requiring a 160 m long column, 20 atm inlet pressure, and unacceptable 8-h or longer analysis for helium).
The peak capacity of a 1D separation can also be increased by reducing the column diameter. However, this also comes with significant difficulties. Thus, doubling the peak capacity of GC–MS without changing the analysis time requires 8-fold narrower columns (12.5 µm i.d. instead of 100 µm i.d.), which leads to a 100-fold larger (worse) minimum detectable concentration (MDC) (minimum analyte concentration), a 16-fold higher pressure, etc.
These considerations highlight the significance of a 10-fold or larger peak capacity increase potentially available from GCxGC without the time increase and worsening MDC.”
In her recent ChromaBLOGraphy posts, Minimizing Phthalate Interferences Using the Rtx-CLPesticides/Rtx-CLPesticides2 Columns and EPA Regulated Phthalates FREE?, my colleague Dan Li pointed out the ubiquitous nature of phthalates in our world because of their widespread use in plastic consumer products, and that phthalates likely have adverse human health effects, including as endocrine disruptors. Any of our blog posts on chemicals create awareness for me as to when those chemicals might pop up at home or work. Recently while attending the 14th International Symposium on Biological and Environmental Reference Materials (BERM 14) in National Harbor, Maryland, USA, I noticed the hotel-supplied shampoo and other bath products advertised “No Phthalates”. Chemical or biological notoriety often creates a market opportunity, as in this case and others (think: non-GMO food, organic or pesticide-free fruits and vegetables, flame retardant-free furniture, 3-MCPD-free soy sauce, etc.). In that regard, putting “No Phthalates” in big letters on a product is inherently different than making a phthalate ingredient declaration via the US Food and Drug Administration’s Fair Packaging and Labeling Act.
Knowing that phthalates are used in shampoos and other personal care products perhaps helps make sense of Penn State University wastewater and Las Vegas Wash water analyses we conducted using GCxGC-TOFMS where phthalates were some of the highest concentration compounds determined. If you need more tips for what phthalates are out there and how to gas chromatograph them, including how to move them away from organochlorine pesticides analyzed by GC-ECD, be sure and check out Dan’s excellent blogs above.
This post is the second of its kind pertaining to LC analysis, all of which are an extension of a series pertaining to “Red Flags” for GC analysis. These are situations and symptoms that tell us in the Tech Service group that something is just not right. As we discussed in the first post, Technical Service “Red Flags” –LC, here are more examples of some “Red Flags” that we sometimes see for HPLC:
Loss in retention:
Customer reports a dramatic loss in retention for all analytes, which elute at or near the void volume.
A secondary complaint with this scenario may also be asymmetrical peak shape, although it may not be noticeable because peaks are often hard to distinguish at all. This is usually caused by a phenomenon known as phase collapse. Here is an example taken from one of our technical articles:
Phase collapse, chain collapse, or dewetting, as it is sometimes called, is the result of exposing a column to a mobile phase with very high aqueous content. A conventional C18 column is not intended to be used with higher than 95% water. What happens is that the mobile phase is so polar that it is repelled by the nonpolar C18 chains and cannot penetrate between the chains. The aqueous solvent will initially fill the pores of the particle, but as soon as the pressure is lowered or the solvent pump is turned off, the water is forced out. The chains then collapse and lay down flat against the surface at this stage. Usually the problem with retention is noticed after the pumps are turned back on and when the next sample is injected thereafter.
The good news is that usually the phase can be rewetted by flushing the column. First make sure any buffer is removed by flushing with mobile phase that contains no buffer salts or modifiers for several column volumes. Then follow that with 100% organic solvent (acetonitrile is preferred). You should able to switch back to your regular mobile phase after that, just make sure that you use no more than 95% water. If your application requires a higher percentage of water in the mobile phase, make sure that you use one of our Aqueous C18 column phases, which include the Ultra Aqueous C18, the Pinnacle DB Aqueous C18 and the Allure Organic Acids columns or a polar embedded column such as our Ultra IBD or Pinnacle DB IBD column.
Visible white residue:
Customer reports visible white residue or particulates coming from the outlet of the column
If this residue does not resemble anything from the samples you have been injecting, then this could be packing material coming out of the column. It is important to STOP the pump flow immediately and disconnect the column outlet so that these particulates do not clog anything downstream of the column in the flowpath. Eventually, this will result in some changes in pressure. Pressure will increase if a clog develops in the flowpath after the material exits the column. If that does not happen (or before it happens), you may actually see a decrease in pressure, deteriorating peak shape and/or a loss in retention of analytes.
The first thing to check when you see this residue is the pH of the mobile phase. If using a pH meter, please confirm that the device is calibrated properly. At some point above a pH of 8.0, the silica may begin to dissolve and when the particles become small enough, they will start coming through the frit at the end of the column. If pH is not the issue, check to see if you have the column installed with the proper direction of flow. Some of our columns are intended to be used with the flow in one direction and cannot be reversed, even for cleaning purposes. This applies to our Pinnacle DB UHPLC (1.9 µm) and our Raptor™ columns. Please look for the arrows on the column that indicate direction of flow to confirm the orientation. Leakage of packing material can also occur if end fittings in the column have been tampered with. For this reason, we do not recommend disassembly of our columns by the customer. If the column is not assembled properly, or the frit has become dislodged, leakage will likely occur. If packing material has escaped from the column, it has been damaged beyond repair and must be replaced.
If, by chance, you find material of this nature is coming instead from the guard cartridge, please stop the pump flow and disconnect the analytical column to minimize damage. Then carefully flush out all of the tubing and fittings before installing a new cartridge.
No peaks at all:
Customer reports no detection of any peaks.
Occasionally we may hear from an analyst with this issue in their lab. For troubleshooting suggestions, please review the earlier blog post Troubleshooting HPLC-Loss in Response for All Analytes. Although very similar to the scenario from the earlier blog post, the following possibilities are more likely when you are seeing no change in baseline at all.:
- Detector is turned off or lamp is not on.
- Incorrect setting of wavelength on detector or attenuation setting is way off.
- A solvent pump is not working properly; flow rate is way too low or no liquid flowing at all.
- No sample is being pulled up for injection or the sample injection (rotary) valve is no longer working.
- Tubing is plumbed incorrectly or there is a very large leak somewhere in the system.
- Sample/standard is not prepared correctly.
If you have ruled out the above possibilities and you still feel the column is at fault, try using the same column in a different HPLC system or try using a different column with the same system. Even when column condition is poor, usually you will see at least a solvent peak or disturbance (“blip”) in the baseline around the time the sample is injected. Switching the column or system should shed some light on the situation.
Please note that it is important to address one thing at a time, so that you can learn the root cause of the problem. If you still need assistance, feel free to contact us at email@example.com.
I hope this has been helpful and thank you for reading.