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New Custom UCMR4 Standard for EPA Method 530 on the Rxi-5Sil MS

We recently stocked custom EPA Method 530 standards made specifically for labs participating in Unregulated Contaminant Monitoring Rule 4 (UCMR4). Quantitative Certificates of Analysis with Data Packs are available for each item. Email csreps@restek.com or phone 800-356-1688 ext. 3 to place your order (items are not available at Restek.com).

EPA Method 530, the Determination of Select Semivolatile Organic Chemicals in Drinking Water by Solid Phase Extraction and Gas Chromatography / Mass Spectrometry was developed on the Rtx-1701, but section 6.12.1 of the method states that “any capillary column that provides adequate capacity, resolution, accuracy, and precision may be used.” 3 of the 4 Method 530 target analytes, butylated hydroxyanisole, o-toluidine, and quinoline have been included in UCMR4; the 4th target analyte, dimethipin, was included in UCMR4 as part of EPA 525.3. It isn’t really clear to me why a new method was made for these compounds instead of relying on EPA Method 525.3. Method 530 uses a 6mL divinyl benzene or polystyrene/divinyl benzene cartridge for extracting 1L where 525.3 has multiple cartridge and disk SPE options. The analysis is a couple minutes faster on the Rxi-5Sil MS under the 525.3 conditions. The selectivity of the two columns are similar, the primary difference being that butylated hydroxyanisole (BHA) is less retained relative to acenaphthene-D10 on the Rxi-5Sil MS. If your lab has limited resources, you should be able to perform both method 530 and 525.3 on one instrument set up for method 525.3.

Cat# 572262 – Method 530 UCMR4 Standard (in Methanol)

Contaminant CAS Registry Number Minimum Reporting Level Standard Concentration
butylated hydroxyanisole (BHA) 25013-16-5 0.03 µg/L 150 µg/mL
o-Toluidine 95-53-4 0.007 µg/L 35 µg/mL
Quinoline 91-22-5 0.02 µg/L 100 µg/mL

 

Cat# 572265 – Method 530 UCMR4 Surrogate Standard (in Methanol)

Contaminant CAS Registry Number Standard Concentration
Quinoline-D7 34071-94-8 500 µg/mL
o-Toluidine-D9 194423-47-7 500 µg/mL

 

Cat# 572266 – Method 530 UCMR4 Internal Standard (in Acetone)

Contaminant CAS Registry Number Standard Concentration
Acenaphthene-D10 15067-26-2 500 µg/mL
Phenanthrene-D10 1517-22-2 500 µg/mL

 

I’ve collected example chromatograms on the Rxi-5sil MS using the same GC conditions that were used for the EPA Method 525.3 UCMR4 standard (Section 10.2.3.1), but with a new SIM program generated using the AutoSIM feature of MSD Chemstation. This was mainly to demonstrate the the two methods can be run on the same GC-MS with the same analytical column.

 

SIM Analysis of EPA 530 UCMR4 Standard Run at Method Reporting Limit (MRL) on the Rxi-5Sil MS

SIM Analysis of EPA 530 UCMR4 Standard Run at 10x the Method Reporting Limit (MRL) on the Rxi-5Sil MS

 

Peaks tR (min) MRL Conc. (µg/mL) 10x MRL Conc. (µg/mL)
1. o-Toluidine-D9 (SUR) 5.64 1.0 1.0
2. o-Toluidine 5.70 0.070 0.070
3. Quinoline-D7 (SUR) 8.22 1.0 1.0
4. Quinoline 8.26 0.20 0.20
5. Butylated hydroxyanisole (BHA) 11.50 0.30 0.30
6. Acenaphthene-D10 (IS) 11.54 1.0 1.0
7. Phenanthrene-D10 (IS) 15.05 1.0 1.0

 

Column Rxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
Sample Method 530 UCMR4 Standard (cat.# 572262)
Method 530 UCMR4 Surrogate Standard (cat.# 572265)
Method 530 UCMR4 Internal Standard (cat.# 572266)
Diluent: Dichloromethane
Injection
Inj. Vol.: 1 µL pulsed splitless (hold 1.0 min)
Liner: 4 mm Single Taper w/Wool (cat.# 23303)
Inj. Temp.: 275 °C
Pulse Pressure: 30 psi (206.8kPa)
Pulse Time: 1.05 min
Purge Flow: 60 mL/min
Oven
Oven Temp.: 70 °C (hold 1.5 min) to 200 °C at 10 °C/min (hold 0 min) to 320 °C at 7 °C/min (hold 3 min)
Carrier Gas He, constant flow
Flow Rate: 1.2 mL/min
Detector MS
Mode: SIM
SIM Program:
Group Start Time (min) Ion(s) (m/z) Dwell (ms)
1 1.543 106, 107, 112, 114 25
2 6.917 102, 108, 129, 136 25
3 9.881 137, 162, 164, 180 25
4 13.297 160, 188 25
Transfer Line Temp.: 280 °C
Analyzer Type: Quadrupole
Source Type: Stainless Steel
Drawout Plate: 6 mm ID
Source Temp.: 280 °C
Quad Temp.: 180 °C
Solvent Delay Time: 1.45 min
Tune Type: DFTPP
Ionization Mode: EI
Instrument HP6890 GC & 5973 MSD

EPA Method 541 on the Stabilwax Column

EPA Method 541 UCMR4 Standard Chromatogram at 10x the Method Reporting Limit on a 30m x 0.25 mm ID x 0.50 µm Stabilwax (cat# 12039).

I recently posted some chromatograms for our new EPA Method 541 UCMR4 Standards, and wanted to focus on the column in a separate post. EPA Method 541 was developed on a 30m x 0.25 mm ID x 0.50 µm wax column from another manufacturer. I used the Stabilwax because it is a relatively low bleed wax column and has similar selectivity to the column used by the EPA. My chromatogram is shown above, while a calibration standard chromatogram taken directly from the method documentation is shown below.

EPA METHOD 541 FIGURE 2 – Reconstructed ion chromatogram (RIC), SIM mode, for calibration standard on a 30m x 0.25 mm ID x 0.50 µm wax column.

There are 2 major differences I wanted to point out:

  1. The Stabilwax column has the same elution profile but it is less retentive at the same phase ratio. The last target compound (chlorobenzene-D5) elutes on the Stabilwax at 8.66 minutes, which is BEFORE the 1st target compound (1,4-Dioxane) elutes on the column used in the EPA Method (10.3 minutes). UCMR4 does not include 1,4-dioxane, so it is not included in the Stabilwax chromatogram. The GC acquisition parameters were the same for both columns, though the detector settings were different because of the SIM window start times. All peaks had eluted on the Stabilwax before the method recommended solvent delay ended.
  2. The bleed is significantly lower on the Stabilwax compared to the calibration standard chromatogram from the EPA method.

New Custom UCMR4 Standards for EPA Method 541

We recently stocked custom EPA Method 541 standards made specifically for labs participating in Unregulated Contaminant Monitoring Rule 4 (UCMR4). Quantitative Certificates of Analysis with Data Packs are available for each item. Email csreps@restek.com or phone 800-356-1688 ext. 3 to place your order (items are not available at Restek.com).

 

Cat# 572263 – Method 541 UCMR4 Standard (in P&T Methanol)

Contaminant CAS Registry Number Minimum Reporting Level Standard Concentration
1-Butanol 71-36-3 2.0 µg/L 2000 µg/mL
2-Methoxyethanol 109-86-4 0.40 µg/L 400 µg/mL
2-Propen-1-ol 107-18-6 0.50 µg/L 500 µg/mL

 

Cat# 572267 – Method 541 UCMR4 Surrogate Standard (in P&T Methanol)

Contaminant CAS Registry Number Standard Concentration
1-Butanol-D10 34193-38-9 50 µg/mL

 

Cat# 572268 – Method 541 UCMR4 Internal Standard (in P&T Methanol)

Contaminant CAS Registry Number Standard Concentration
Chlorobenzene-D5 3114-55-4 50 µg/mL

The analyte concentrations make it easy to prepare calibration levels based on the minimum reporting level (MRL) required by the UCMR4 program. I’ve included example chromatograms collected at the MRL and 10x the MRL using the GC conditions taken from EPA Method 541. The SIM conditions were generated using the AutoSIM feature of MSD Chemstation version D.03.

SIM Analysis of EPA 541 UCMR4 Standard Run at Method Reporting Limit (MRL) on the Stabilwax

SIM Analysis of EPA 541 UCMR4 Standard Run at 10x the Method Reporting Limit (MRL) on the Stabilwax

Peaks tR (min) MRL Conc. (µg/mL) 10x MRL Conc. (µg/mL)
1. 2-Propen-1-ol 6.94 0.013 0.13
2. 1-Butanol-D10 (SUR) 7.25 0.25 0.25
3. 1-Butanol 7.38 0.050 0.50
4. 2-Methoxyethanol 7.97 0.010 0.10
5. Chlorobenzene-d5 8.66 0.25 0.25

 

Column Stabilwax, 30 m, 0.25 mm ID, 0.50 µm (cat.# 10638)
Sample Method 541 UCMR4 Standard (cat.# 572263)
Method 541 UCMR4 Internal Standard (cat.# 572268)
Method 541 UCMR4 Surrogate Standard (cat.# 572267)
Diluent: Dichloromethane
Injection
Inj. Vol.: 1 µL pulsed splitless (hold 0.50 min)
Liner: 4 mm Single Taper w/Wool (cat.# 23303)
Inj. Temp.: 200 °C
Pulse Pressure: 10 psi (68.9kPa)
Pulse Time: 0.55 min
Purge Flow: 100 mL/min
Oven
Oven Temp.: 30 °C (hold 0.5 min) to 110 °C at 10 °C/min (hold 0 min) to 200 °C at 25 °C/min (hold 6 min)
Carrier Gas He, constant flow
Flow Rate: 0.9 mL/min
Detector MS
Mode: SIM
SIM Program:
Group Start Time (min) Ion(s) (m/z) Dwell (ms)
1 6.000 39, 55, 57, 58 25
2 7.153 41, 43, 46, 50, 55, 56, 63, 64 25
3 7.685 45, 47, 58, 76 25
4 8.292 76, 117, 119 25
Transfer Line Temp.: 200 °C
Analyzer Type: Quadrupole
Source Type: Stainless Steel
Drawout Plate: 6 mm ID
Source Temp.: 230 °C
Quad Temp.: 150 °C
Solvent Delay Time: 6.0 min
Tune Type: BFB
Ionization Mode: EI
Instrument HP6890 GC & 5973 MSD

 

New Custom UCMR4 Standard for EPA Method 530 on the Rtx-1701

We recently stocked custom EPA Method 530 standards made specifically for labs participating in Unregulated Contaminant Monitoring Rule 4 (UCMR4). Quantitative Certificates of Analysis with Data Packs are available for each item. Email csreps@restek.com or phone 800-356-1688 ext. 3 to place your order (items are not available at Restek.com).

Cat# 572262 – Method 530 UCMR4 Standard (in Methanol)

Contaminant CAS Registry Number Minimum Reporting Level Standard Concentration
butylated hydroxyanisole (BHA) 25013-16-5 0.03 µg/L 150 µg/mL
o-Toluidine 95-53-4 0.007 µg/L 35 µg/mL
Quinoline 91-22-5 0.02 µg/L 100 µg/mL

 

Cat# 572265 – Method 530 UCMR4 Surrogate Standard (in Methanol)

Contaminant CAS Registry Number Standard Concentration
Quinoline-D7 34071-94-8 500 µg/mL
o-Toluidine-D9 194423-47-7 500 µg/mL

 

Cat# 572266 – Method 530 UCMR4 Internal Standard (in Acetone)

Contaminant CAS Registry Number Standard Concentration
Acenaphthene-D10 15067-26-2 500 µg/mL
Phenanthrene-D10 1517-22-2 500 µg/mL

 

The suggested analytical column for EPA Method 530, the Determination of Select Semivolatile Organic Chemicals in Drinking Water by Solid Phase Extraction and Gas Chromatography / Mass Spectrometry, is the Rtx-1701, though “any capillary column that provides adequate capacity, resolution, accuracy, and precision may be used.” 3 of the 4 Method 530 target analytes, butylated hydroxyanisole, o-toluidine, and quinoline have been included in UCMR4; dimethipin, the 4th target analyte, was included in UCMR4 as part of EPA 525.3. I’ve included example chromatograms collected at the method reporting limit (MRL) and at 10x the MRL below. The GC inlet, oven and detector settings were taken directly from the method, and the AutoSIM function in MSD Chemstation version D.03 was used to generate the SIM analysis windows.

SIM Analysis of EPA 530 UCMR4 Standard Run at Method Reporting Limit (MRL) on the Rtx-1701

SIM Analysis of EPA 530 UCMR4 Standard Run at 10x the Method Reporting Limit (MRL) on the Rtx-1701

Peaks tR (min) MRL Conc. (µg/mL) 10x MRL Conc. (µg/mL)
1. o-Toluidine-D9 (SUR) 8.426 1.0 1.0
2. o-Toluidine 8.481 0.070 0.70
3. Quinoline-D7 (SUR) 10.622 1.0 1.0
4. Quinoline 10.641 0.20 2.0
5. Acenaphthene-D10 (IS) 13.45 1.0 1.0
6. Butylated hydroxyanisole (BHA) 14.25 0.30 3.0
7. Phenanthrene-D10 (IS) 17.318 1.0 1.0

 

Column Rtx-1701, 30 m, 0.25 mm ID, 0.25 µm (cat.# 12023)
Sample Method 530 UCMR4 Standard (cat.# 572262)
Method 530 UCMR4 Surrogate Standard (cat.# 572265)
Method 530 UCMR4 Internal Standard (cat.# 572266)
Diluent: Dichloromethane
Injection
Inj. Vol.: 1 µL pulsed splitless (hold 1.0 min)
Liner: 4 mm Single Taper w/Wool (cat.# 23303)
Inj. Temp.: 275 °C
Pulse Pressure: 20 psi (206.8kPa)
Pulse Time: 1.05 min
Purge Flow: 60 mL/min
Oven
Oven Temp.: 60 °C (hold 1.0 min) to 290 °C at 10 °C/min (hold 1 min)
Carrier Gas He, constant flow
Flow Rate: 1.0 mL/min
Detector MS
Mode: SIM
SIM Program:
Group Start Time (min) Ion(s) (m/z) Dwell (ms)
1 3.50 106, 107, 112, 114 25
2 9.55 102, 108, 129, 136 25
3 12.08 162, 164 25
4 14.02 137, 180 25
4 15.92 160, 188 25
Transfer Line Temp.: 280 °C
Analyzer Type: Quadrupole
Source Type: Stainless Steel
Drawout Plate: 6 mm ID
Source Temp.: 280 °C
Quad Temp.: 180 °C
Solvent Delay Time: 3.5 min
Tune Type: DFTPP
Ionization Mode: EI
Instrument HP6890 GC & 5973 MSD
Notes The EPA Method 530 UCMR4 Standard analyte concentrations vary to simplify preparing ICAL levels based on the minimum method reporting levels.

New Custom UCMR4 Standard for EPA Method 525.3

We recently stocked a custom EPA Method 525.3 standard made specifically for labs participating in Unregulated Contaminant Monitoring Rule 4 (UCMR4). Quantitative Certificates of Analysis with Data Packs are available for each item. Email csreps@restek.com or phone 800-356-1688 ext. 3 to place your order (items are not available at Restek.com)

Cat# 572261 – Method 525.3 UCMR4 Standard – Eight Pesticides and One Pesticide Manufacturing Byproduct (in Methanol)

Contaminant CAS Registry Number Minimum Reporting Level Standard Concentration
alpha-hexachlorocyclohexane 319-84-6 0.01 µg/L 10 µg/mL
chlorpyrifos 2921-88-2 0.03 µg/L 30 µg/mL
dimethipin 55290-64-7 0.2 µg/L 200 µg/mL
ethoprop 13194-48-4 0.03 µg/L 30 µg/mL
oxyfluorfen 42874-03-3 0.05 µg/L 50 µg/mL
profenofos 41198-08-7 0.3 µg/L 300 µg/mL
tebuconazole 107534-96-3 0.2 µg/L 200 µg/mL
total permethrin (42% cis- & 58% trans-) 52645-53-1 0.04 µg/L 40 µg/mL
tribufos 78-48-8 0.07 µg/L 70 µg/mL

 

The analyte concentrations make it easy to prepare calibration levels based on the minimum reporting level (MRL) required by the UCMR4 program. I’ve included example chromatograms collected at the MRL and 10x the MRL using the GC conditions taken from section 10.2.3.1 of EPA Method 525.3 version 1.0. The SIM conditions were generated using the AutoSIM feature of MSD Chemstation version D.03.

SIM Analysis of EPA 525.3 UCMR4 Standard Run at Method Reporting Limit (MRL)

SIM Analysis of EPA 525.3 UCMR4 Standard Run at 10x Method Reporting Limit (MRL)

Peaks tR (min) MRL Conc. (µg/mL) 10x MRL Conc. (µg/mL)
1. 1,3-Dimethyl-2-nitrobenzene (SUR) 7.43 1.0 1.0
2. Acenaphthene-D10 (IS) 11.54 1.0 1.0
3. Ethoprop 13.29 0.030 0.30
4. Alpha-BHC 14.06 0.010 0.10
5. Dimethipin 14.54 0.20 2.0
6. Pentachlorophenol-13C6 (IS) 14.65 2.0 2.0
7. Phenanthrene-D10 (IS) 15.05 1.0 1.0
8. Chlorpyrifos 17.09 0.030 0.30
9. Profenofos 19.34 0.30 3.0
10. Tribufos 19.49 0.070 0.70
11. Oxyfluorfen 19.57 0.050 0.50
12. Tebuconazole 21.7 0.20 2.0
13. Triphenyl phosphate (SUR) 21.8 1.0 1.0
14. Chrysene-D12 (IS) 22.63 1.0 1.0
15. cis-Permethrin 24.98 0.017 0.17
16. trans-Permethrin 25.16 0.023 0.23
17. Benzo[a]pyrene-d12 (SUR) 26.68 1.0 1.0

 

Column Rxi-5Sil MS, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13623)
Sample EPA Method 525.3 PAH IS Mix (cat.# 32547)
Pentachlorophenol-13C6 (cat.# 32548)
EPA Method 525.3 Surrogate Standard (cat.# 32549)
Method 525.3 UCMR4 Standard (cat.# 572261)
Diluent: Ethyl Acetate
Injection
Inj. Vol.: 1 µL pulsed splitless (hold 1.0 min)
Liner: 4 mm Single Taper w/Wool (cat.# 23303)
Inj. Temp.: 275 °C
Pulse Pressure: 30 psi (206.8kPa)
Pulse Time: 1.05 min
Purge Flow: 60 mL/min
Oven
Oven Temp.: 70 °C (hold 1.5 min) to 200 °C at 10 °C/min (hold 0 min) to 320 °C at 7 °C/min (hold 3 min)
Carrier Gas He, constant flow
Flow Rate: 1.2 mL/min
Detector MS
Mode: SIM
SIM Program:
Group Start Time (min) Ion(s) (m/z) Dwell (ms)
1 1.546 77, 134 25
2 9.501 162, 164 25
3 12.423 97, 126, 139, 158 25
4 13.666 109, 181, 183, 219 25
5 14.288 53, 54, 274, 276 25
6 14.856 160, 188 25
7 16.087 97, 197, 199 25
8 18.205 57, 63, 97, 139, 169, 208, 252, 339, 361 25
9 20.652 77, 83, 125, 169, 250, 326 25
10 22.212 236, 240 25
11 23.813 163, 183 25
12 25.886 132, 164 25
Transfer Line Temp.: 280 °C
Analyzer Type: Quadrupole
Source Type: Stainless Steel
Drawout Plate: 6 mm ID
Source Temp.: 280 °C
Quad Temp.: 180 °C
Solvent Delay Time: 1.45 min
Tune Type: DFTPP
Ionization Mode: EI
Instrument HP6890 GC & 5973 MSD

How can I use Raptor columns and the EXP Direct connect holder with stainless steel fittings?

Some customers may find themselves with this scenario. You have an HPLC instrument with existing stainless steel tubing and fittings and you are getting ready to install a Raptor column. The end of your tubing may very well look like the one shown above. Here are some questions you might have.

 

Can I attach this stainless steel fitting directly to a Raptor or other Restek LC column?

Yes, this is possible to do with a Raptor column or any of our Restek LC columns. To form the best seal and avoid leakage, it is preferred to start with a nut and ferrule that is not already swaged (tightened down). To obtain the best seal, insert the tubing until it bottoms out and then tighten the fitting. The difference between using stainless steel fittings and PEEK type fittings is discussed further here in the FAQs section of our website: http://www.restek.com/Pages/faq_lc#pkd30

 

Can I attach this stainless steel fitting directly to an EXP Direct Connect Guard holder (catalog #25808)?

Again, yes this is possible, and the same conditions apply that I mentioned for connecting directly to the column. Particularly if you intend to operate up to 600 Bar, it would be best to start with a nut and ferrule that is not already swaged to obtain the best seal.

 

Is it better to use some other type of fitting to connect to a Raptor column or EXP Direct Connect holder?

We prefer to use an EXP fitting to connect a Raptor column or the EXP guard holder for several reasons.

Reason # 1:  Usually you can tighten the EXP fittings by hand and will not need a wrench. Not only is this just a whole lot easier, but it results in a longer lifetime for the end fitting of the Raptor column, or longer lifetime for the end fitting on the EXP holder (called a “holder cap”) in the figure below.

 

Reason #2: The EXP fittings will work with either PEEK tubing or stainless steel tubing.  If using stainless steel tubing, EXP2 fittings (shown below), may also be used, but these are tightened with a driver nut that comes with the fitting when you purchase it.

Reason #3:  As mentioned in the above link to the FAQs, the EXP fittings do not require permanent swaging. This means that unless you use a wrench to tighten it, the ferrules are removable and can be reused.   Please note that ferrules used with EXP2 fittings are also somewhat reusable, even though a more permanent seal is created that is rated up to 20, 000 psi.

 

How would I go about using an EXP fitting to attach my column or guard holder if I already have the stainless steel nut and ferrule swaged to the end of my tubing?

This is the tricky question and there are several different ways at least to do this.

 

 

 

 

 

 

 

 

 

 

Option #1: Replace this section of tubing with PEEK tubing and add EXP fittings to both ends. Particularly if you don’t plan to operate above 600 bar on this instrument, the PEEK tubing will be easier to work with and it is preferred by most analysts.  You will find our selection of PEEK tubing here on our website:
http://www.restek.com/catalog/view/944

EXP fittings can be found here:

http://www.restek.com/catalog/view/37868

 

 

Option #2: Since we do not recommend cutting the stainless steel tubing, the swaged nut and ferrule cannot be easily removed. If for some reason, you need to continue using stainless steel tubing, you can just replace the length of stainless steel tubing with a new one. We sell lengths of ss tubing here on our website: http://www.restek.com/catalog/view/46845  You will need to check your instrument manual to see which tubing ID it recommends for this instrument model. In many cases, this length of tubing is 0.17 mm or 0.007 inches ID. If you plan to use a tubing assembly that already has stainless steel fittings on it, make sure you start with one that is not swaged yet on the end that goes to the column (so you can remove the nut and ferrule).  You can then use either EXP or EXP2 fittings to attach the column. Most likely, you can use the same type of fitting at the other end also.

 

 

 

 

 

 

 

 

Option #3: This is the most creative and versatile option. If you want to keep the existing stainless steel tubing and fittings in place, but prefer to use an EXP (or EXP2) fitting to attach your column or guard holder, this is what you need to do:

Attach a zero dead volume union, such as our catalog #20147, or something similar with 10-32 threads (most typically used for LC) to the existing stainless steel fitting. The bore size of the union should be as close to the ID of your tubing as possible. If the union comes with metal nuts and ferrules, you will not need them.

Obtain an EXP coupler, catalog #25940. Attach one end to the union and the other end to the Raptor column or the EXP Direct Connect guard holder (catalog # 25808). Once tightened down, you should be ready to go.

 

I hope you have found this post helpful. Thank you for reading.

 

 

 

LC Columns: High-Pressure Survival of the Fittest!

As an R&D Chemist charged with developing the perfect packing method for new LC columns, one of the challenges is finding a packing method that results in a column which not only passes QC specifications but also passes our arduous lifetime analysis. During lifetime testing our columns are subjected to 1,000 continuous injections at 12,000 PSI (a pressure far higher than what most LC operators will ever need- it’s the equivalent of being under 27,680 feet of water!).

You may ask yourself, “Why bother actually doing 1,000 injections at high pressure? Couldn’t one just run the column at high pressures or flow rates and see lifetime degradation?” What many people don’t realize is that the principal cause of column degradation over time are variations in pressure that occur with each individual injection. These pressure ‘pulses’ are unique to each make and model of HPLC or UHPLC, and are controlled by the structure and mechanics of the instrument.

Finding a rugged packing method that can withstand this kind of punishment isn’t easy – and proved especially challenging when developing the packing methods for our 1.8 µm Force columns. Once we finally hit on the right method I was exceedingly (embarrassingly) excited and jumped at the chance to pit our new ‘survivors’ against the competition. Utilizing our lifetime test and two replicates of each column, we assessed Restek Force C18 (1.8 µm, 2.1 x 50 mm) column performance in comparison to similar dimension, particle, and stationary phase columns from two well-known competitors:

From the graph above, you can see that Restek and Brand 1 columns showed excellent ruggedness, and maintained ≥94% of their original efficiency over the course of the lifetime test. Brand 2 column performance suffered however, with one column steadily dropping in efficiency by the end of the test, and the other maintaining efficiency for 250 injections before plummeting, after which peak integration became impossible due to peak splitting.

At Restek one of our core values is pride in our work. Seeing the results of our hard work in R&D and knowing that we can offer these excellent LC columns to customers gives me great pride to be a Restek R&D chemist.

Thanks for checking out my first blog post!

p.s. Our other dimensions and phases of Force columns are just as rugged as the ones I’ve shown you above. If you want to see another one of my beautiful graphs and learn more about Force please check out our overview here: Force Performance LC Columns Overview .

The Clog Blog

Clogging and over pressuring are definitely problematic for Liquid Chromatography columns. Back in 2005 John Dolan’s sage advice to chromatographers to make an LC column last forever was simple, “Never open the box!” As John pointed out, this is not practical and I have to agree. But as an LC column manufacturer I have seen remarks from customers regarding clogging as though it was it was a flaw in the column. Unfortunately, there is not really anything that we do when we manufacture a column that would cause one column clog faster than another. Typically, if a column clogs, it is due to particulate matter that is larger than the pores of the column frit getting stuck in the frit and not allowing the flow to pass through. As obvious and simple as this sounds, it can still be overlooked.

To understand LC column clogging, it is important to know how a modern LC column is built. It is actually pretty straight-forward. A metal tube has an endfitting attached to the bottom. This fitting has two purposes. It provides a means to attach your LC’s tubing to the column and it holds a porous filter or frit in place. Consider the frit as a semipermeable membrane. It has pores that allow the liquid mobile phase to pass through, but are not big enough to allow the column packing particles to pass through. I’ll talk more about the frit later, as it is very important to clogging.

Back to building the column. Once one of the endfittings, the bottom one, is attached to the tube, the packing material, or bonded particles (think of spherical silica of generally uniform diameter, coated with C18 ligands) are mixed into a solvent solution to make a slurry, and pumped into the column tube under pressure. Obviously, the solvent from the slurry passes through the frit that was fixed to the end of the tube with the endfitting and the particles are stopped by the frit. The slurry is pumped into the tube until the tube is filled to the top with particles. You can then remove the tube from the packing pump and affix another frit and endfitting to the open end of the tube. Now the particles that were packed into the tube cannot get out either end and you have an LC column. The particles are packed so tightly into the tube that there is no extra space between the packing material and the frit. Any extra space would be called a void and a void in a column will spoil your day. It can result in problems that will affect the efficiency and peak shape in your separations. For this reason, we recommend that the user never try to take off the endfittings from their column. You will never get a tight fit like a column manufacturer can. So if you take the fittings off the column it is now pretty much scrap. Even though I simplified the packing process here, it is actually a very precise process that takes a great deal of time to perfect. Different particles behave differently as do different phases, column lengths, and different internal diameters. Better to leave it to the professionals.

Click on image for a larger view.

Back to frits. They are so important to clogging because just as they are the right size to let liquid through but not the particles in the column, they allow your HPLC mobile phase pass though when you connect the column to your HPLC. Now keep in mind the particles inside an LC column are very small. A typical HPLC column has 3 to 5 micron particles. If you look on the internet you will find that an average human red blood cell is about 5 µm in diameter. Now that is similar to a relatively large HPLC column particle. UHPLC columns typically have particles that are less than 2 µm in diameter. Remember the pores in the column frit need to be smaller than the particles inside the column. And when a column manufacturer says that a column has 3 or 5um particles know that that is an average. Silica is a natural product so there is a distribution of particle sizes inside that column. A 3um column could have, for instance, particles ranging from 2 to 4um in it. It is very typical for a column manufacturer to use a 2um average pore size frit for 5um particle columns and a 0.5um average frit for 3um or smaller particle columns. I say average for the frit pore sizes because of how they are made. Frits are typically sintered metal. Sintering is process of heating up small uniform metal bits to near melting point and then pressing them so that they fuse together. The spaces that are left between the fused metal bits are the pores. By being layered they create channels or pores in the material. Some may be larger or smaller than others but the process is controlled and repeatable to make a more or less uniform material. This porous metal material is cut into small discs that HPLC column manufacturers use as frits.

So when you are injecting your sample into the column consider what is in your syringe. Was your sample soil or water, blood or urine, or maybe a plant extract? Do you know what size particles are in your sample? Will they pass through the column frit or will they get stuck in the pores? There are some very easy ways to be sure that you won’t clog the frit on your column and give it a nice long operating lifetime. The easiest thing to do is prefilter your sample. If you know that your column has a 0.5um frit, you should prefilter it through a membrane that is smaller than 0.5um, perhaps a 0.45um or 0.2um syringe filter or filter vial. The better you can filter your sample (translation the smallest filter you can use) the better. Maybe you think that your sample is free of particles. Try centrifuging it. If you can see anything at the bottom of the tube, you have a dirty sample. But let’s face it, the human eye could never see a 1um or even a 10um particle in a solution. Of course there are other ways to protect your column’s precious frit. A precolumn disposable frit for example. Again choose one that is, ideally, smaller than the frit in your column. Or a guard column. A good quality guard column will have 2 frits on it just like your column. If the precolumn frit or guard column get clogged, just throw them away and get a new one that is what they are for. Your column is protected.

Your support team at Restek can help you with several different options to help you keep your column from clogging. Remember, if you want your column to last forever, keep it in the box. But if you want your column to maintain its operating pressure under use for a long time, you now know how it works so think before you inject.

Utilizing Restek Reference Standards for Residual Solvent Testing

Restek manufactures several different residual solvent standards.  These can be used for testing according to USP <467>, the EP monograph, or other residual solvent assays, such as medical cannabis testing.   The calculations involved for the use of these standards has led to questions for our technical team.  This post should help provide guidance for their usage.  I’ll use USP <467> in my example calculations, but they can be applied to other methodology.

Since USP <467> is a limit test, each residual solvent has a concentration limit based upon its potential risk to human health.   For that reason, not all compounds are at the same concentration in our reference standards.  As an example, Benzene has a limit of 2 ppm for the Class 1 Residual Solvents.  Catalog #36279 contains benzene at 10 mg/mL.  This solution would be what USP refers to as Residual Solvent Mixture RS.

According to the monograph, you are to do the following:

Class 1 Standard Stock Solution— [Note—When transferring solutions, place the tip of the pipet just below the surface of the liquid, and mix.] Transfer 1.0 mL of USP Class 1 Residual Solvents Mixture RS to a 100-mL volumetric flask, previously filled with about 9 mL of dimethyl sulfoxide, dilute with water to volume, and mix.

 Using Catalog #36279

10,000 µg/mL x 1.0 mL / 100 mL = 100 µg/mL

 Transfer 1.0 mL of this solution to a 100-mL volumetric flask, previously filled with about 50 mL of water, dilute with water to volume, and mix.

 100 µg/mL x 1.0 mL / 100 mL = 1.0 µg/mL

 Transfer 10 mL of this solution to a 100-mL volumetric flask, previously filled with about 50 mL of water, dilute with water to volume, and mix.

 1.0 µg/mL x 10 mL / 100 mL = 0.1 µg/mL Class 1 Standard Stock Solution

Class 1 Standard Solution— Transfer 1.0 mL of Class 1 Standard Stock Solution to an appropriate headspace vial containing 5.0 mL of water (place the tip of the pipet just below the surface of the liquid for dispensing), apply the stopper, cap, and mix.

0.1 µg/mL x 1.0 mL / 6.0 mL = 0.0167 µg/mL

Note: Final volume = 6 mL (1 mL of Class 1 Stock Solution + 5 mL Water)

This solution is the one analyzed by headspace GC/FID.  It is less important to consider the 0.0167 µg/mL concentration of benzene than it is to know that there is 0.1 µg of benzene in the vial.   This 0.1 µg of benzene is the amount partitioned into the gaseous headspace and injected into the GC

1 mL of 0.1 µg/mL = 0.1 µg (benzene)

With your drug substance or excipient, you are to do the following according to USP:

 Test Stock Solution— Transfer about 250 mg of the article under test, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

 250 mg / 25 mL = 10 mg/mL

 Test Solution— Transfer 5.0 mL of Test Stock Solution to an appropriate headspace vial, add 1.0 mL of water, apply the stopper, cap, and mix.

Since this is the solution analyzed by headspace GC/FID, it is more important to know how much article is present and not the concentration.

10 mg/mL x 5 mL = 50 mg of article tested

The limit for benzene is 2 ppm (0.002 µg/mg), which happens to be the concentration you would find by having 0.1 µg of benzene in your 50 mg of article.

0.1 µg (benzene) / 50 mg (article) = 0.002 µg/mg (2 ppm)

You can do the same math for any of the other compounds in the Class 1 Mix.

The dilution scheme for Class 2 Solvents is similar to the Class 1 Solvents.   Since Class 2 Solvents are considered not as toxic as the Class 1, the limits are much higher.  Since the allowable limit is so much greater, Restek has accounted for the first dilution with the Class 2 Solvents.    This is in Catalog #36012.  For the Class 2 Solvents, I will use acetonitrile as an example.

Following USP and the calculations outlined above, the procedure would be:

2050 µg/mL x 1.0 mL / 100 mL = 20.5 µg/mL

20.5 µg/mL x 1.0 mL/ 6.0 mL = 3.417 µg/mL or 20.5 µg of acetonitrile

20.5 µg (acetonitrile) / 50 mg (article) = 0.41 µg/mg (410 ppm) = limit for acetonitrile

I hope you find this helpful.

Some notes about Fats and Oils, cis/trans-Fatty acids and our latest re-launched RT 2560

I recently posted a Blog regarding some notes about Fats and Oils, FAMES and our Famewax column, which can be found here. In Food Industry the challenge may be a bit heavier, if the amount of Trans Fatty Acids is requested.

There is scientific consensus that trans fats intake has a negative effect on human health: more specifically, consumption of trans fats has a negative impact on blood cholesterol levels and increases the risk of heart disease more than any other macronutrient compared on a per-calorie basis; the risk of dying from heart disease is 20 – 32% higher when consuming 2% of the daily energy intake from trans fats instead of consuming the same energy amount from carbohydrates, saturated fatty acids, cis monounsaturated fatty acids and cis polyunsaturated fatty acids (*Mozaffarian D et al., 2009, Health effects of trans-fatty acids: experimental and observational evidence, European Journal of Clinical Nutrition 63(S2): p. S5-S21)
Although it has to be noted that the intake of Industrial Trans Fatty Acids (ITFAs) has decreased in the past years in the EU, they are still excessively present in some diets and particular population groups in the EU, such as low-income citizens in the UK, university students aged 18 to 30 years in Croatia or generally citizens of this age range in Spain are identified as risk groups with an intake higher than 1% of daily energy intake (which is the recommended value by WHO).

Therefore some European Countries have taken action and have introduced a legal limit of IFTAS in Food, starting with Denmark in 2003, followed by some other countries like Austria, Hungary and Latvia.  Voluntary self-regulation industrial measures have been implemented in Belgium, Germany, the Netherlands, Poland, the UK and Greece. Legal measures limiting in this area exist also in Switzerland, Iceland, Norway and the US.

This said, there is obviously a lack of homogeneity in the whole EU in relation to limitation of IFTAs in food, what is causing problems in an effective functioning of the internal EU market (e.g. protection of consumers’ health). Therefore SANTE (EU Directorate General of Health and Food Safety) has started a new “Initiative to limit industrial trans fats intakes in the EU”, which may lead to an increasing challenge in Food characterization. The final SANTE statement is supposed to be given during 3rd quarter of 2017.

Measuring the Fatty Acid composition of Food, including IFTAs, is challenging and normally follows the EN ISO 12966-2015, Part 1 to 4 (Animal and vegetable fats and oils –Gas chromatography of fatty acid methyl esters), which is also ground base of some national method developments as given in DGF C-VI 10a/11a in Germany

For us, as one of the leading  GC separation column producer, in particular the ISO 12966­4:2015 “Animal and vegetable fats and oils – Gas chromatography of fatty acid methyl esters – Part 4: Determination by capillary gas chromatography” is the most important part, giving some recommendations of suitable columns to be used.

Chapter 5 (Apparatus) indicates as a suitable Capillary column “fused silica capillary 100 m and 0,25 mm i.d. coated with 100 % cyanopropylsilicone stationary phase to a thickness of 0,20 µm.”

Due to its high polarity this column is not easy to be manufactured, as many users may have recognized in the past. This column cannot be manufactured as a bonded version. Until now, this results in some draw back, like change of polarity during lifetime (having changes in retention times) and in some production inconsistencies, recognized as differing batch to batch reliability. This is due to phase chemistry and could be seen all around commercial available products.

Restek scientists therefore have started an initiative to overcome most of the mentioned issues. Having all production processes under control, starting with the manufacturing of raw fused silica and the in-house production of high end polymers to achieve the best possible stationary phase, we have improved a lot of the manufacturing processes, which gave us the opportunity to implement tighter controls on specifications to guarantee low bleed and a more stable baseline. This initiative leads to more consistency in the product and much more reliability for your measurements.

This sounds like a huge step forward in characterizing the fat composition of Food, especially regarding the analysis of the mentioned ITFAs. Some of the impressive results can be seen here.

But as always, the best proof is to check these results by yourself. Ask your regional Restek Sales Representative about this column and make your own decision.

HJM