PROGETTO UNIVA 2013 Journal Club Pietro Gareri, MD, PhD Geriatra ASP Catanzaro Lamezia Terme 3 Luglio 2013 Drug-induced parkinsonism (DIP) was recognized in the early 1950s as a commoncomplication of antipsychotic therapy; initially considered to be present in 4 - 40%of patients treated with the first neuroleptics
Supelco Ionic LiquidsThe Dawning of a New Era in GC Phase Technology • Liquid Chromatography • Sample Handling • Gas Chromatography The evolution of GC Phase Technology
from Substituted Polysiloxane Polymers
and Polyethylene Glycols in the 1950's,
to Bonded Phases in the 1980's, and on
to our new Ionic Liquid phases today.
See you at the Conference! Visit us on the web at sigma-aldrich.com/thereporter Market Segment Manager Dear Col eague,
Conferences and exhibitions are an excel ent opportunity for vendors and conferees to share information on their latest developments and research. It also gives vendors an opportunity to get feedback from customers on new "Contributed Articles" are submitted product needs and requirements. Every year Supelco attends over 30 tradeshows worldwide, some very small by our customers based upon their and focused in very specific areas of analytical chemistry like the ASMS Conference and others very large and work with Sigma-Aldrich products. diverse like PITTCON. We encourage you to submit articles Just like you, we can't attend every tradeshow, but we are trying to make it easier for you to stay current with describing your work for consideration in future publications. our new developments and to get information on what we present at many of the tradeshows that we attend. Below are a few of the ways we can help you keep up with the most recent information we are presenting at the upcoming conferences. We would like for you to attend the tradeshows and stop by our booth, but if you can't attend then you can: Table of Contents l Request a CD containing oral and poster presentations from PITTCON® 2008. Liquid Chromatography l Visit our website (sigma-aldrich.com/analytical-events) to see the most up-to-date list of tradeshows and conferences that we are attending and find recently presented materials.
l Contact Technical Service or your local sales specialist to get more information on new products and recent publications.
Upcoming major tradeshows where you can visit us:
HPLC 2008 International Symposium Int'l Symposium on Capil ary Chromatography Riva Del Garda, Italy American Chemical Society Mtg Gulf Coast Conference Gas Chromatography American Association of Pharma Scientists Eastern Analytical Symposium We wil also be at other smal er, more focused shows. For more information on these shows, visit our website at sigma-aldrich.com/analytical-events. We hope to see you at one of the upcoming tradeshows! You can also find information on upcoming local technical seminars on the events page.
Market Segment Manager Reporter is published 5 times a year by Supelco Marketing, 595 North Harrison Road, Bel efonte, PA 16823-0048.
Accelerating Customers' Success through Leadership in Life Science, High Technology and Service
Supelco Patented Ionic Liquid GC Phase Technology
Leonard M. Sidisky and Michael D. Buchanan
Figure 1. Modifiable Components of a Dicationic Phase With a few exceptions, just two types of columns are typical y used in gas chromatography (GC). The first type includes columns that contain a substituted polysiloxane polymer phase, the origin of which can be traced back to the 1950's and the very birth of the GC technique. The second type is comprised of columns coated with a [1,9-di(3-vinyl-imidazolium) nonane bis(trifluoromethyl) sulfonyl imidate] G004214, G004216, G004215 polyethylene glycol phase, a phase that has remained virtual y unchanged nearly as long as GC has been of ionic liquids permits numerous opportunities for practiced. With the successful use of ionic liquids as viable modification. As shown in Figure 1, the components that Gas Chromatography GC stationary phases, analysts will be able to perform can be modified include the cation, linkage and anion. previously unthought of separations. l Both dicationic (shown) and polycationic ionic liquids have been shown to make suitable GC phases.
The Revolution: Ionic Liquid GC Phases
l The choice of cations evaluated thus far includes Chromatographic characteristics: Ionic liquids are a imidazolium (shown), phosphonium, and pyrrolidini- um. Plans exist for evaluating others. The cations class of non-molecular ionic solvents with low melting may be the same, but do not necessarily need to be.
points. These liquids are unique combinations of cations l Modifications to the linkage include changing type and anions. The practical use of ionic liquids as GC and/or length. For example, an alkane (shown) or stationary phases has long been desired because of polyethylene glycol, or some other type, of various lengths might be used. If the ionic liquid is polyca- tionic, the types and lengths of the linkages may be 1. The ability to remain liquids over wide temperature the same, but they can be different.
ranges, expanding the GC column operating l The number of anion candidates is also large. The temperature range compared to traditional initial work has included the use of bis(trifluoromethyl) (US and Canada only) stationary phases (substituted polysiloxane sulfonyl imidate [nTf2-] (shown) and trifluoromethyl polymers or polyethylene glycols).
sulfonate [triflate], which has shown promise in 2. Very low volatility, providing low column improving peak symmetry.
bleed, stable retention times, long column l Additional y, further modifications of the cation or life, and increased maximum temperatures.
linkage, (such as the addition of pendant groups, 3. Highly polar nature, expanding the polarity derivatization, or chiral characteristics) can be scale upward.
explored. Short alkanes, vinyl groups (shown), and 4. Novel selectivity, al owing application-specific use.
hydroxyl groups are a few choices that have been successful y used for cation modification.
Previous work with ionic liquids as GC phases focused l Other possibilities exist, such as bonding, crosslinking, on monocationic ionic liquids, which did not exhibit the blending ionic liquids, or doping into existing non- desired chromatographic characteristics specified above. ionic GC phases.
Prof. Daniel Armstrong (University of Texas at Arlington) Experimentation has shown that modification to any has expanded on this work, showing that dicationic and single component, even a slight modification in linkage polycationic ionic liquids as GC phases exhibit desired length, can be used to achieve a desired chromatographic benefit. Through further characterization, it is hoped that the relationships between the effects on phase character-istics / chromatographic performance caused by modifica- With the successful use of ionic liquids / 814-359-3441 technical service: 800-359-3041 tions of each of these components can be predicted, as viable GC stationary phases, leading to the rapid development of phases/columns with analysts wil be able to perform targeted selectivities. Supelco R&D chemists are actively previously unthought of separations. involved in discovering these cause and effect relationships.
Sigma-Aldrich/Supelco is the first-to-market with this Phase modification: Whereas the chemical structures new, innovative, and patented (US 2008/0027231 A1; other of existing GC phases al ow limited modification (changing patents pending) technology, developed in conjunction with the pendant group on polysiloxane polymers or adjusting Prof. Daniel Armstrong (University of Texas at Arlington).
the length of polyethylene glycols), the chemical structure (continued on page 4) dering: 800-247-6628 or
Figure 2. BTEX and n-Alkanes on the SLB-IL100 Figure 3. Rapeseed Oil FAMEs on the SLB-IL100 column: SLB-IL100, 30 m x 0.25 mm I.D., 0.20 µm (28884-U) column: SLB-IL100, 30 m x 0.25 mm I.D., 0.20 µm (28884-U) 1. Myristic (C14:0) det.: FID, 250 °C det.: FID, 250 °C 2. Palmitic (C16:0) carrier gas: helium, 26 cm/sec @ 110 °C carrier gas: helium, 30 cm/sec @ 180 °C 3. Stearic (C18:0) injection: 0.1 µL, 300:1 split injection: 1 µL, 100:1 split 4. Oleic (C18:1n9c) liner: 4 mm I.D., split, cup liner: 4 mm I.D., split, cup 5. Linoleic (C18:2) sample: NEAT mixture containing v sample: Rapeseed oil FAME mix, 6. Linolenic (C18:3) percentages of each component 5 mg/mL total FAMEs in 7. Arachidic (C20:0) methylene chloride 8. cis-11-Eicosenoic 9. Behenic (C22:0) 10. Erucic (C22:1) 11. Lignoceric (C24:0) Gas Chromatography (continued from page 3) di-, and tri-unsaturated fatty acids ranging in carbon length The SLB™-IL100: The First Phase in the Line
from C14 to C24. The elution of C18:3 after C20:0 and The SLB-IL100 is the first commercial offering in this new C20:1 is typical y observed with highly polar phases.
line of novel columns, those that utilize ionic liquid phase Based on the rapeseed oil elution pattern and other chemistry. The SLB-IL100 column has a polarity/selectivity characterizations, it has been determined that the SLB- roughly equivalent to that of the traditional TCEP phase, IL100 phase is virtual y equivalent in polarity/selectivity to and exemplifies some of the desired characteristics that the TCEP phase, currently one of the highest polarity/ ionic liquids are predicted to possess. Namely, a higher selectivity GC phases.
maximum temperature compared to non-ionic liquid columns with similar polarity/selectivity. Specifical y, the Outlook for Ionic Liquid GC Phase Technology
SLB-IL100 has a 230 °C maximum temperature, resulting The patented and successful use of ionic liquids as viable from the robustness and low volatility of the phase, GC stationary phases heralds in a new and exiting chapter whereas traditional TCEP columns with equivalent polarity/ in GC phase technology. Now analysts wil be able to selectivity have a 140 °C maximum temperature. The perform previously unthought of separations with the SLB-IL100 column is expected to broaden the range of potential to go way beyond applications possible using applications that can be performed on highly polar columns.
traditional phases. For example, an ionic liquid phase with a polarity/selectivity similar to that of Carbowax® 20M, but Example Applications on the SLB-IL100
with a maximum temperature over 300 °C, is just one of BTEX and n-Alkanes
many possibilities currently being investigated. Look for Figure 2 il ustrates the separation of benzene, toluene, additional Supelco ionic liquid phases to be introduced in ethyl benzene, and the xylene isomers (BTEX) in the the coming months. This is truly an exciting time in GC presence of C11 and C13 n-alkanes. The high polarity/ phase development! selectivity of the ionic liquid phase results in the elution of toluene after C13 at 110 °C. This is desirable because the SLB-IL100 Specifications aliphatic fraction of gasoline consists of n-alkanes up to Phase: non-bonded; 1,9-di(3-vinyl-imidazolium) nonane C13. Therefore, the quantitation of aromatics in products bis(trifluoromethyl) sulfonyl imidate Temp. Limits: Subambient to 230 °C based on gasoline (such as mineral spirits) requires a column with a polarity/selectivity able to separate the aromatic fraction from the aliphatic fraction. As shown, + Featured Products
the SLB-IL100 has this necessary polarity/selectivity.
Rapeseed Oil FAMEs
SLB-IL100 Fused Silica Capillary Column
Figure 3 il ustrates the separation of the fatty acids 30 m x 0.25 mm I.D., 0.20 µm (analyzed as FAMEs) found in a rapeseed oil sample. Rapeseed oil contains a variety of saturated and mono-,
Improve GC Reproducibility by Using
FocusLiner™ Inlet Liners
Robert F. Wallace
The wool plug can be easily dislodged without Figure 2. Tailing Solvent Peak – Wool Plug in the chromatographer's Incorrect Position knowledge. As shown in Poor sample reproducibility observed by chromatogra- Figure 1, a common cause phers from one consecutive injection to another may be of wool plug displacement an indication that small variations in the injection volume within the liner is that have occurred. Placing a small plug of either glass or repeated injections quartz wool inside an inlet liner has historical y been used progressively move the to overcome this. However, this procedure does have Gas Chromatography wool plug until no further distinct drawbacks. FocusLiner inlet liners are specifical y contact with the needle is designed to exhibit the benefits of using a wool plug made. A sudden change in without the drawbacks.
the inlet pressure, like changing the septum, can With a FocusLiner inlet liner, the also result in the move- chromatographer can be assured that the Figure 3. Sharp Solvent ment of the wool plug. Peak – Wool Plug in Relocation of the wool wool plug is always in the correct position plug from the correct position can be character- The Problem with Wool Plugs in Traditional Liners
ized by excessive tailing of In addition to preventing non-volatile material from the solvent peak, as shown (US and Canada only) entering the column, a wool plug exhibits two benefits in Figure 2. As shown in that assist in reducing injection volume variability. 1) The Figure 3, sharp solvent increased surface area facilitates the maximum vaporiza- peaks are only observed tion of the sample. 2) Any droplets formed on the outside when the wool plug is in of the needle are wiped off. Both of these benefits require the correct position to that the needle tip penetrate the wool plug. Therefore, wipe the needle tip.
the position of the wool plug in the injection liner is critical. Unfortunately, there is no guarantee that once a Overcoming the Drawbacks
liner is instal ed in the injector that the wool plug will stay FocusLiner inlet liners utilize an innovative design that in the correct position.
overcomes the drawbacks observed with the use of wool plugs in traditional inlet liners. With FocusLiner inlet liners, Figure 1. The Problem – Wool Plug in Traditional Inlet Liner the wool plug is held in position by two tapered sections. As shown in Figure 4 (page 6), these tapered sections secure the wool plug in the correct position, even after repeated injections and exposure to sudden pressure changes. With a FocusLiner inlet liner, the chromatogra- / 814-359-3441 technical service: 800-359-3041 pher can be assured that the wool plug is always in the THE PROBLEM:
correct position. This will ensure that the needle tip penetrates the wool plug, wiping any residual liquid preventing needle wiping or sample sample from the needle tip while providing sufficient surface area for maximum volatilization of the sample.
The effect on sample precision (measured as %RSD) Inlet Liner caused by the position of the wool plug in the liner was (continued on page 6) dering: 800-247-6628 or withdrawal. As shown in Figure 2, a tailing solvent peak Figure 4. The Solution – FocusLiner Inlet Liner may interfere with the quantitation of peaks that elude shortly after the solvent peak. As shown in Figure 3, the correct position of the wool plug results in sharp solvent peaks and more accurate quantitation of peaks that elude Two tapered sections (A) secure
shortly after the solvent peak.
the wool plug (B) in the correct
position to ensure improved Poor reproducibility and severe tailing may be observed if the needle tip is not wiped during injections. As the (continued from page 5) sample is being delivered from the syringe, droplets will measured. A 4 mm I.D. traditional inlet liner with the wool form that wet the syringe needle tip. The volume of plug moved to the center was evaluated against a 4 mm sample that remains on the needle tip varies from I.D. FocusLiner inlet liner. Another frequently used split Gas Chromatography injection to injection. The key to the improved reproduc- liner was also evaluated. This liner design substitutes the ibility provided by the FocusLiner inlet liner is the proper wool plug with a sintered glass frit, which can be either positioning of the wool plug in the liner, al owing the fixed or removable. In this experiment a 4 mm I.D. fixed needle tip to be wiped. The use of a FocusLiner inlet liner frit liner was used.
provides precise, accurate, and reliable sample injections, As shown in Figure 5, when the wool plug is moved to resulting in improved reproducibility. the center of the traditional inlet liner, %RSD values are in the 8-10% range. 1. Technical Article TA-0043-A, FocusLiner: Improve GC Accuracy and Reproducibility Figure 5. %RSD of Different Impressively, the Focus- 10 Fold, SGE (www.sge.com) 2. Technical Article TA-0004-A, 0.2% RSD's? It's Now a Reality with SGE's FocusLiner, Wool Plug Positions Liner inlet liner was able SGE (www.sge.com) to achieve %RSD values 3. R.F. Wal ace, Supelco, TheReporter, August 2006; Volume 24.4: 11 w/wool in center of liner for the same probe w/fixed sintered glass frit compounds in the 0.2% FocusLiner
range! This is up to 50 Description
times lower than those measured with the Split/Splitless, 78.5 x 6.3 x 4.0 mm traditional inlet liner.
Split/Splitless, 78.5 x 6.3 x 4.0 mm, single taper PerkinElmer® AutoSystem™ and Clarus
The fixed sintered glass Split/Splitless, 92 x 6.2 x 4.0 mm frit liner is also unable to Split/Splitless, 92 x 6.2 x 4.0 mm, single taper Shimadzu®14/15A/16 with SPL-14 Injector
match the precision Split/Splitless, 99 x 5.0 x 3.4 mm provided by the FocusLin- Split/Splitless, 99 x 5.0 x 3.4 mm, single taper Shimadzu 17A with SPL-17 Injector
er inlet liner. This result is Split/Splitless, 95 x 5.0 x 3.4 mm not surprising as a key Split/Splitless, 95 x 5.0 x 3.4 mm, single taper element to achieving Varian® 1075/1077 Injector
Split, 72 x 6.3 x 4.0 mm good sample reproducibil- Split, 72 x 6.3 x 4.0 mm, single taper ity is the needle tip being Varian 1078/1079 Injector
Split/Splitless, 54 x 5.0 x 3.4 mm, single taper wiped during injection. Therefore, liners with fixed or Varian CP-1177 Injector
removable frits can only be used with limited success.
Split/Splitless, 78.5 x 6.3 x 4.0 mm Split/Splitless, 78.5 x 6.3 x 4.0 mm, single taper 1. Al of these FocusLiner inlet liners are packs of 5 and are packed with quartz wool. Additional pack sizes can be viewed at our website Sample accuracy is also a critical factor in providing confidence in sample quantitation. Peak areas for probe compounds using the FocusLiner inlet liner were found to ! Related Information
be, on average, 25% higher than a liner where the wool plug is positioned incorrectly.
Our full line of FocusLiner inlet liners can be viewed online at sigma-aldrich.com/focusliner. For more information on inlet Solvent peak tailing is also observed if the wool plug is liners, request the Capil ary GC Inlet Liner Selection Guide, incorrectly positioned, caused by slow vaporization near T196899 (BBB) and the poster Selecting the Appropriate Inlet the cool septum cap as the needle is wiped during Liner, T404081 (HCH).
Improving the Chiral Separation of Dorzolamide
A Case Study of Chiral Analytical Services
they are remarkably stable and effective in reversed phase, normal phase, polar organic, and polar ionic chromato- In the mid 1980s and early 1990s, several pharmaceuti- graphic modes without memory effects. The CHIROBIOTIC cal companies began to explore the development of phases mainly rely on strong anionic or cationic binding, carbonic anhydrase inhibitors for the topical treatment of hydrogen bonding, and p-p complexation to achieve glaucoma. Dorzolamide, a water soluble sulfonamide separation of various enantiomers (3). Conversely, the possessing two chiral centers, emerged as a product of CYCLOBOND phases rely on inclusion as the retention these research efforts (1).
mechanism. Inclusion complexing arises due to apolar segments of chiral molecules becoming attracted to the Figure 1. Structures of Dorzolamide and its Enantiomer apolar cyclodextrin cavity. While apolar segments may occupy the inside of the cavity, more polar segments of the analyte may interact through dipole-dipole interactions, hydrogen bonding, and steric interactions at the mouth of the cavity, al owing the cyclodextrin phases to distinguish Liquid Chromatography between isomers differing in stereochemistry. The CYCLO-BOND phases thrive in reversed phase and polar organic modes, and some (CYCLOBOND I 2000 RN, SN, DMP, and DNP) are also compatible with normal phase mode (4). A sample consisting of a 1:1 mixture of dorzolamide Although the development of enantiomerical y pure hydrochloride and its enantiomer [(4R, 6R)-4-(Ethylamino- chiral drugs such as dorzolamide has recently proven extremely beneficial to the treatment of various ailments, sulfonamide 7,7-dioxide, monohydrochloride)] (dorzolamide (US and Canada only) manufacturers face the obstacle of chiral separation. hydrochloride Related Compound A (2)) was tested in a Previous efforts focusing on the chiral separation of chiral screening protocol employing six of the Astec CHIRO- dorzolamide from its undesired chiral enantiomer [(4R, 6R)- BIOTIC and CYCLOBOND phases most likely to give positive results: CHIROBIOTIC V2 (Vancomycin), CHIROBIOTIC T (Teicoplanin), CHIROBIOTIC TAG (Teicoplanin Aglycone), ride)] have been complicated. The chromatographic mode CYCLOBOND I 2000 (b-cyclodextrin), CYCLOBOND I 2000 of separation described in the 2006 United States Pharma- DNP (b-cyclodextrin, 3,5-Dinitrophenyl carbamate) CYCLO- copeia, for instance, involves a derivatization of racemic BOND I 2000 HP-RSP (b-cyclodextrin, High Performance dorzolamide with chiral reagent (S)-(-)-a-methylbenzyl isocyanate prior to separation on a non-chiral silica phase Although the development of enantiomerical y (2). Elimination of this derivatization step would both pure chiral drugs has proven extremely beneficial to conserve time and decrease expenses associated with the the treatment of various ailments, manufacturers synthesis and analysis of dorzolamide. face the obstacle of chiral separation. In an effort to simplify the chiral separation of dorzol- R,S-hydroxypropyl ether) (25 cm x 4.6 mm I.D., 5 µm particle amide from its undesired enantiomer without prior deriva- size). Mobile phases encompassing reversed-phase (70:30, / 814-359-3441 technical service: 800-359-3041 tization, a recent study employed Astec CHIROBIOTIC™ 20 mM ammonium acetate, pH 4.0:acetonitrile), and polar and CYCLOBOND™ columns in a chiral screen of the two ionic (100:0.1:0.1, methanol:acetic acid:triethylamine) dorzolamide enantiomers. chromatographic modes of operation were applied to the The Astec CHIROBIOTIC phases consist of macrocyclic CHIROBIOTIC phases. The CYCLOBOND phases were glycopeptides linked covalently to a silica surface by five screened in the aforementioned reversed-phase mobile covalent bonds. These phases possess broad selectivity and phase as wel as a mobile phase referred to as polar organic can differentiate between smal variability in chemical mode (95:5:0.3:0.2, acetonitrile:methanol:acetic acid: structure, making them valuable in the separation of a wide triethylamine). Screening was executed on the Waters 2690 array of chiral molecules. Unlike most other chiral phases, Separations Module utilizing a Waters 996 Photodiode Array dering: 800-247-6628 (continued on page 8) (continued from page 7) TAG, the CHIROBIOTIC T showed no separation in both Detector (UV at 220 and 254) and Waters Empower reversed-phase and polar ionic modes of operation.
Acquisition Software (2002 Version).
Unlike the CHIROBIOTIC phases, the CYCLOBOND Subsequent to initial screening, positive results were columns in the screen showed no positive results. The confirmed and optimized on the Agilent 1100 series HPLC dorzolamide enantiomers were unretained on both utilizing a VWD detector with a UV wavelength of 254 nm. CYCLOBOND I 2000 and CYCLOBOND I 2000 HP-RSP and Table 1 summarizes the results of the primary screen. unresolved on the CYCLOBOND I 2000 DNP in reversed- The summary table shows evidence of enantiomeric phase. Polar organic mode produced no discernible peaks selectivity observed on both the CHIROBIOTIC V2 (V2) on the CYCLOBOND phases.
and CHIROBIOTIC TAG (TAG) in polar ionic mode and on Because the V2, under polar ionic conditions, produced the V2 in reversed-phase. Near baseline resolution was the best resolution of the dorzolamide enantiomers in the revealed on the V2 in polar ionic mode, while only partial primary screen, it was selected for optimization purposes. separation was observed on both the TAG in polar ionic Consecutive attempts at optimization, including a decrease mode and the V2 in reversed phase. Unlike the V2 and in flow rate from 1.0 mL/min down to 0.25 mL/min and a Table 1. Primary Screen Summary of Dorzolamide and its Enantiomer on Astec CHIROBIOTIC and CYCLOBOND phases Mobile Phase
Liquid Chromatography 70:30, 20 mM NH OAc (pH 4.0):ACN 100:0.1:0.1, MeOH:HOAc:TEA Partial Separation 70:30, 20 mM NH OAc (pH 4.0):ACN Partial Separation 100:0.1:0.1, MeOH:HOAc:TEA 70:30, 20 mM NH OAc (pH 4.0):ACN 100:0.1:0.1, MeOH:HOAc:TEA CYCLOBOND I 2000 70:30, 20 mM NH OAc (pH 4.0):ACN CYCLOBOND I 2000 95:5:0.3:0.2, ACN:MeOH:HOAc:TEA CYCLOBOND I 2000 HP-RSP 70:30, 20 mM NH OAc (pH 4.0):ACN CYCLOBOND I 2000 HP-RSP 95:5:0.3:0.2, ACN:MeOH:HOAc:TEA CYCLOBOND I 2000 DNP 70:30, 20 mM NH OAc (pH 4.0):ACN CYCLOBOND I 2000 DNP 95:5:0.3:0.2, ACN:MeOH:HOAc:TEA decrease in temperature from 25 °C down to 10 °C, succeeded in increasing resolution; however baseline Figure 3. Analysis of Dorzolamide and its Enantiomer Using pH Adjusted Buffered Mobile Phase 5:95, resolution was not quite achieved. The buffer salt 20 Ammonium Formate (pH 4.0):Methanol ammonium formate was employed to sharpen the peaks column: CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 µm particles and enhance resolution. As seen in Figure 2, when mobile phase: 5:95, 20 mM ammonium formate (pH 4.0):methanol coupled with the mobile phase 0.05 w% ammonium flow rate: 1.0 mL/min formate in methanol, the aforementioned temperature det.: UV at 254 nm and flow rate changes produced near baseline resolution. sample: 1.0 mg/mL in methanol Ultimately, Figure 3 depicts that slight modification of the 2. [(4R, 6R)-4-(Ethylamino-5,6-dihydro-6-methyl- ammonium formate mobile phase to include the addition 4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide, monohydrochloride)] of water and pH adjustment (5:95, 20 mM ammonium formate, pH 4.0:methanol) gave baseline resolution of the dorzolamide enantiomers with the original temperature and flow rate (25 °C, 1.0 mL/min). Figure 2. Analysis of Dorzolamide and its Enantiomer Using Buffered Mobile Phase 0.05 w% Ammonium Formate in Methanol on the CHIROBIOTIC V2 Liquid Chromatography column: CHIROBIOTIC V2, 25 cm x 4.6 mm I.D., 5 µm particles mobile phase: 0.05 w% ammonium formate in methanol flow rate: 0.25 mL/min efficiency. For this reason, procedures involving chiral det.: UV at 254 nm separation without prior derivatization have become much sample: 1.0 mg/mL in methanol more attractive to the scientific community than lengthy and dated derivatization procedures.
2. [(4R, 6R)-4-(Ethylamino-5,6-dihydro-6-methyl- 4H-thieno[2,3-b]thiopyran-2-sulfonamide 7,7-dioxide, monohydrochloride)] 1. J. Borras et. al. Bioorg. Med. Chem. 1999, 7, 2397-2406.
(US and Canada only) 2. United States Pharmacopeia, 29th rev,; United States Pharmacopeial Convention: Washington, DC. 2005; pp 756-757.
3. CHIROBIOTIC Handbook, 5th ed.; T406120, JEV, Supelco, 595 North Harrison Road, Bel efonte, PA 16823.
4. CYCLOBOND Handbook, 7th ed.; T406119, JEU, Supelco, 595 North Harrison Road, Bel efonte, PA 16823.
+ Featured Products
Of the six columns screened, the best resolution of the Astec CHIROBIOTIC V2 Chiral HPLC Column dorzolamide chiral enantiomers, conclusively, was observed 25 cm × 4.6 mm I.D., 5 µm particles on the V2 stationary phase under polar ionic mode condi-tions. The vancomycin phase contains two ionic sites, making ! Related Information
it especial y good for the separation of both acidic and basic For more information on custom chiral screening, method molecules. Decreasing the pH to 4.0 enhanced the ionic development, or chiral purification, please visit our website at interactions between the secondary nitrogen attached to one sigma-aldrich.com/astec, or contact Supelco Technical Service chiral center of the analyte and the carboxylic acid groups of at 800-9-01 (US and Canada only), 81-9-01, or email / 814-359-3441 technical service: 800-359-3041 the V2 stationary phase. Conformational differences cause one enantiomer to have a slightly stronger affinity to the stationary phase than the other, thus, enhancing chiral Did you know.?
separation of the dorzolamide enantiomers.
Supelco now offers Chiral Screening Services to assist
Like the aforementioned separation of the dorzolamide customers in analytical method development and purifica- enantiomers, chiral separations that may be achieved tion. The screening service consists of an initial screen of without extra derivatization steps conserve considerable Astec chiral columns, method optimization, and purification amounts of time. Since decreasing time ultimately conserves of enantiomers. Enantiomers are identified as (+) or (-) money, modern industries constantly focus efforts on using the Chiralyser optical rotation detection system. dering: 800-247-6628 seeking abbreviated procedures aimed at increasing their Rapid, Sensitive, General-Purpose Cleaning Validation
Using Ascentis® Express HPLC Columns
related analytes comes at the expense of run time and is The fol owing was generated by an outside source using not needed in cleaning validation.
Sigma-Aldrich products. Technical content provided by: This work was undertaken to investigate the use of S. Bannister, M. Talbott, F. Hanciles
rapid gradients using recently introduced FCP columns on Xcelience LLC, Tampa, FL conventional instrumentation in the development of general-purpose methods for cleaning validation. The Verification of the removal of drug residue from multi- benefits include high sensitivity and reductions in the time product manufacturing equipment is required by GMP needed to set up and run the method. regulations and the suitability of applied analytical Resolution, limits of detection and quantitation, and run methods is judged with a combination of sensitivity, time in HPLC analyses are improved by reducing the width selectivity, and because the release of equipment is of eluted bands. Contributions to bandwidth include both Liquid Chromatography dependent - speed. The FDA does not set quantitative column (particle size, packing structure and resistance to acceptance specifications, but the commonly used limit is mass transfer in the stationary and mobile phases) and based on not more than 0.1% of a dose carried over into extracolumn volumes (injection, unswept and tubing). a single dose of the next product. Translation of this into Columns packed with 5 µm ful y porous particles have an analytical limit combines the total product contact been the standard for conventional HPLC for twenty-five area, the mass (or volume) of product contacting the years. Smal er-particle packings (3 µm) have been surface, the mass (or volume) of each dose unit, the available almost as long and offer higher efficiency (lower sampled area, the rinse volume and the fraction of the band dispersion) on conventional instrumentation, but rinse sample used for analysis. The requisite limits are require higher pumping pressures due to lower bed commonly measured in ng/mL of injected sample. permeability. Efficiency can be further increased by the The ubiquity of HPLC in drug analysis makes it an use of particles smal er than 3 µm but only with the use of attractive choice for cleaning validation. Methods instrumentation optimized with respect to both pressure qualified for cleaning validation are often adaptations of and extra-column effects.
drug-substance methods. The original methods are Supelco has recently introduced reverse-phase packings capable of determining the drug and its related impurities, based on 2.7 µm silica particles in which a 0.5 µm layer of but the ability to simultaneously measure multiple closely 90-Å porous silica has been deposited onto a 1.7 µm solid Figure 1. Fused-Core Structure of Ascentis Express Compared to Total y Porous Particles Ascentis Express Particle
Totally Porous Particle spherical core (Figure 1). Advantages of columns packed The high resolving power of gradient elution in the with these particles include high efficiency, lower analysis of closely related substances is the result of the backpressure due to a very narrow particle size distribu- reduction of peak width as a band moves through the tion, and smal er efficiency losses with increasing velocity column. The back of the band is accelerated by the due to improved mass-transfer kinetics in the shal ow stronger solvent. A broad gradient will elute a wide range porous layer. The narrow particle size distribution al ows of substances and a steep gradient will elute them quickly.
the use of larger pore column frits, which combined with the greater stability of the packed bed should produce longer column lifetimes in routine use.
To judge the utility of Ascentis Express columns in cleaning validation, an Agilent 1100 component system Figure 2. Acidic and Neutral Drug Panel with standard components (including a 10 mm/13 µL flow cel ) was used to develop a short gradient separation using column: Ascentis Express C18, 10 cm x 4.6 mm I.D. (53827-U) mobile phase A: water with 0.1% phosphoric acid Ascentis Express C18, 10 cm x 4.6 mm for each of two mobile phase B: acetonitrile with 0.1% phosphoric acid panels: eight acidic or neutral drugs (AN) and six basic drugs flow rate: 1.76 mL/min 1. Hydrochlorothiazide (9) (B). For each separation, the flow rate was 1.76 mL/min, det.: UV at 215 nm 2. Chlorthalidone (2) 3. Prednisolone (2) detection was at 215 nm, and 100 µL injections were made gradient: Min %A %B 4. Pravastatin (4) 5. Carbamazepine (2) of aqueous solutions representing the final equipment 6. Diclofenac (14) rinse. The separations are shown in Figures 2 & 3. Limits of Liquid Chromatography 7. Ibuprofen (15) 8. Progesterone (2) detection (ng/mL) are listed next to each analyte in Figures These separations demonstrate the capabilities of Ascentis Express columns on conventional, robust, instru- mentation in rapid analyses of multiple drugs at low ppb levels suitable for development as methods for cleaning validations in multiproduct manufacturing facilities.
(US and Canada only) + Featured Products
Ascentis Express Columns
Figure 3. Basic Drug Panel column: Ascentis Express C18, 10 cm x 4.6 mm I.D. (53827-U) mobile phase A: water with 0.05M potassium phosphate and 0.1% TEA and 0.6% OSA-Na at pH = 2.9 mobile phase B: acetonitrile flow rate: 1.76 mL/min 2. Dipyridamole (29) det.: UV at 215 nm 3. Propranolol (12) 4. Haloperidol (8) gradient: Min %A %B 5. Amlodipine (29) 6. Fluoxetine (3) ! Related Information
/ 814-359-3441 technical service: 800-359-3041 For more information on Ascentis Express columns, request T407044 (JHD) or visit sigma-aldrich.com/express dering: 800-247-6628 or Ascentis® Express HILIC HPLC Columns
A breakthrough in HPLC Analysis of Polar Molecules on Ascentis Express HILIC and C18 columns: Ascentis Express HILIC, 10 cm x 2.1 mm I.D., 2.7 µm particles (53939-U) column performance. Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 µm particles (53823-U) mobile phase: 10:90; 100 mM ammonium formate, pH 3.0 with concentrated formic acid:acetonitrile flow rate: 0.4 mL/min Key Benefits of HILIC 1. Acenaphthene, 80 µg/mL in mobile phase l Retention of highly polar det.: UV at 254 nm 2. Adenosine, 35 µg/mL in mobile phase Liquid Chromatography analytes like metabolites injection volume: 1 µL 3. Cytosine, 75 µg/mL in mobile phase l Increased MS sensitivity l Orthogonal selectivity to C18 Ascentis Express HILIC Ascentis Express C18 Author Visits Supelco Booth at PITTCON Professor Eugene F. Barry (University of Massachusetts at Lowel ), co-author of Columns for Gas Chromatography: Performance and Selection, stopped by the Supelco booth at PITTCON. This 2007 Wiley book, ISBN 978-0-471-74043-8, should prove to be a great educational tool for those new to GC as well as an invaluable resource for those experienced in GC.
Dr. Eugene Barry with Product Manager Mike Buchanan (left) and V.P. of Research & Development Mark Robil ard (right) at PITTCON® 2008.
LC-MS Mobile Phase Additives - Tips & Tricks
pH provided by the salt that permits both positive and negative ion mode detection are issues of concern (6). LC-MS is becoming a routine analytical tool in Sodium Adduct Formation
research and industrial laboratories. The demand on sensitivity, specificity and speed of analysis requires use Formation of alkali adduct is associated with decrease in of high purity chemicals for sample preparation, mobile sensitivity. When adduct formation tendency is strong, phase and post-column additives. Additives are used to addition of smal and defined amounts of sodium ions suppress unwanted signals to selectively enhance the (mostly pre-column) can help to obtain uniform and stable signal of particular compounds in a mixture, for example, molecular ions for detection in LC-MS (7). In addition to glycosidic species in a mixture of peptides. Salts can sensitivity, stability and perhaps specificity, of the molecular suppress ionization in ESI sources. ion are also important. The ability to form alkali adducts is useful for quantifying certain classes of molecules and for Acids – The Most Common Additives
selectively enhancing the LC-MS signals. However, their true Volatile, low molecular weight organic acids like formic benefit, particularly that of sodium ion needs further studies.
and acetic acid are commonly used as additives in LC-MS Sigma-Aldrich offers a wide range of high purity mobile phase. Their primary advantage is that they improve additives for LC-MS applications in addition to pure ionization and resolution of a wide range of molecules (1). CHROMASOLV solvents and ready-to-use blends. Our Overcoming the TFA Suppression Effects
offering includes the most commonly used acids, bases, volatile salts and a sodium source (see Featured Products The ionization-supressing effects of trifluoroacetic acid below). All products are of high purity, usual y puriss p.a., (TFA) can be partly overcome by addition of other LC-MS and are tested for LC-MS applications. compatible organic acids, like formic or propionic acid (2). Mobile phases for HPLC of proteins and peptides usual y contain TFA to control the pH and improve peak shape and 1. Emmert J., Analytix, 2006, no.2, 8.
(US and Canada only) 2. Emmert J. and Rueck A, Analytix, 2006, No. 3, 16. resolution. TFA enhances retention by ion pairing with the 3. "Eliminate TFA and Improve Sensitivity of Peptide Analysis by LC-MS" Supelco Ap- peptide and improves peak shape by reducing silanol plication Note 168 (T302168).
4. Apffel A., Fisher S., Goldberg G, Goodley P.C., Kuhlmann F.E., J. Chromatography, interactions (3). However, TFA has adverse effects on MS A, 1995, 712, 177-190.
detection. Its high surface tension prevents efficient spray 5. Wang G., Cole R.B., J. Am. Soc. Mass Spectrom., 1996, 7(10), 1050-1058.
formation and TFA ions in the gas phase ion-pair with the 6. Emmert J. and Leitner A, Analytix, 2006, No. 4, 9.
7. Emmert J. and Waelti T, Analytix, 2006, No. 5, 6.
peptide basic group suppressing their ionization and reducing the MS signal (4,5). + Featured Products
The Neutral Salts
Pkg. Size Cat. No.
The neutral volatile salts, ammonium acetate and Eluent Additives for LC-MS
ammonium formate, offer a much broader influence on Trifluoroacetic acid, puriss* p.a. analyte separation and ionization than do acids (6). Their Trifluoroacetic acid, puriss p.a. Formic acid, puriss p.a. use, of course, is dictated by the particular LC-MS Acetic acid, puriss p.a. Propionic acid, puriss p.a. Ammonium formate, puriss p.a. Ammonium acetate, puriss p.a. It may be necessary under certain circumstances to use Sodium citrate tribasic dihydrate, puriss p.a. Ammonium bicarbonate, puriss p.a. more neutral conditions, either because the analytes are Ammonium hydroxide solution 25%, puriss p.a. / 814-359-3441 technical service: 800-359-3041 Triethylamine, puriss p.a. sensitive to acids or do not exhibit optimal resolution at low LC-MS CHROMASOLV® Blends
pH. When acids are not suitable, volatile salts like ammo- Water with 0.1% ammonium acetate nium formate or acetate may be the additives of choice. Methanol with 0.1% ammonium acetate Acetonitrile with 0.1% ammonium acetate However, limited solubility of the salt in organic solvents, Acetonitrile with 0.1% formic acid Selection of LC-MS Solvents and Blends
changing pH value during a gradient and the mildly acidic 2-Propanol CHROMASOLV LC-MS Water with 0.1% formic acid and 0.01% TFA Acetonitrile with 0.1% formic acid and 0.01% TFA * "puriss" quality grade is defined as >98.5% assay, <0.1% ash, and specifica- tion n + 0.001, d + 0.001 with no extraneous color and a homogeneous For more information, request KCT on the attached postcard appearance. "p.a." or pro analysis denotes a product with guaranteed trace impurity levels and/or suitability for the indicated analytical application.
dering: 800-247-6628 and visit our website: sigma-aldrich.com/chromasolv Blood Fatty Acid Assessment
Kits for Sample Collection and
on the fatty acid content in the blood samples. Subse- Derivatization for GC Analysis
quently, it helps the care providers in development and Monitoring fatty acid profile in blood is important for application of adequate preventive dietary strategies for optimizing fat intake and managing dietary plans for patients. Blood samples col ected as a small drop from the We offer a complete line of products special y designed fingertip can be analyzed to provide sufficient data for for analysis of fatty acids. For more information, please such an assessment (1).
Sigma-Aldrich offers kits for convenient col ection of blood drops, their storage, shipment, and processing the 1. Marangoni F., Colombo C., Gal i C., Anal. Biochem, 2004, 326, 267-272.
samples for fatty acid analysis via gas chromatography. A processing kit contains the derivatization reagent: hydrogen chloride-methanol 1.25 M solution. This reagent is used to derivatize the blood sample for an accurate and Description
effective GC analysis. The treatment al ows efficient Blood Col ection Kit evaluation of the fatty acid status (3-n and 6-n polyun- Includes blood col ection dipsticks, desiccant packs, foil-barrier ziplock bags, 50 mL BHT solution and complete instructions. saturated fatty acids). Enough for 100 tests. Derivatization Kit These kits al ow efficient sample col ection and Includes methanolic HCl solution (1.25M), saturated KCl solution, distil ed water and working instruction sheet. Enough for 100 tests. processing for quick col ection of analytical information Analyses of Fatty Acids A specially designed complete product line from Supelco Solvents & Reagents l Capillary GC Columns l Chemical Standards
Vials, Syringes & Labware l Solid Phase Extraction l Technical Information
For al your FAME analytical needs visit sigma-aldrich.com/fame
For additional information, cal technical service at 800-359-3041 / 814-359-3041
l Products sorted by GC, HPLC,
Chiral and TLC techniques
l Reagents also listed by "Application"
l Vials, syringes and other useful items
for derivatization reactions
l Up-to-date application information
New! Derivatization Brochure
Listing over 400 Derivatization Reagents
To order your free copy either go to sigma-aldrich.com/derivatization, cal 800-359-3041 (US and Canada)
or 814-359-3041, email [email protected] or request KDI on the attached card.
(US and Canada only) SPE Phases Catered to Your Compounds SupelMIP™ SPE consists of MIP Phases & Applications molecularly imprinted polymers
for extraction of trace analytes l Clenbuterol in urine from complex matrixes.
l Triazines in water l b-agonists and b-blockers in tissue, urine and wastewater l Chloramphenicol in milk, plasma, l NNAL and TSNAs in urine l Riboflavin in milk / 814-359-3441 technical service: 800-359-3041 Reduce Ion-suppression l Achieve Lower Detection Limits
Superior Selectivity l Minimal Method Development
To learn more about SupelMIP SPE, or to request a sample pack, please visit sigma-aldrich.com/supelmip
dering: 800-247-6628 or Supelclean™ Sulfoxide SPE for the Extraction of
PCBs and other Aromatic Compounds in Oil
Researchers have found that dimethylsulfoxide (DMSO) liquid-liquid extraction (LLE) is an effective means of The fol owing was generated by an outside source using separating PCBs (aromatic hydrocarbons) from aliphatic Sigma-Aldrich products. Technical content provided by: hydrocarbons (transformer oil) prior to GC-MS analysis (4). Although effective, LLE is often tedious, time consuming Toshiro Kaneko, Charles Mi, Michael Ye, An Trinh
and not greatly amenable to higher throughput applica- 1. AIST, National Metrology Institute of Japan, Tsukuba, 305-8563, Japan tions. Based on the same extraction principles behind 2. Supelco, 595 N. Harrison Rd., Bel efonte, PA, 16823, USA the DMSO LLE approach, we discuss the utility of a sulfoxide-bonded SPE stationary phase towards the extraction of PCBs from transformer oil. Using this new SPE phase, we are able to achieve quantitation levels Polychlorobiphenyls (PCBs) were once heavily used as an below 0.5 ppm (mg/kg). indestructible coolant and insulating fluid in transformer and capacitor oils, and also as a stabilizing additive for a Supelclean Sulfoxide SPE – How it Works
variety of products such as lubricating oils, hydraulic fluids, Supelclean Sulfoxide SPE consists of a patent pending flame retardants, paints and adhesives. However, because silica-bonded sulfoxide (-SO) phase (Figure 1). The of their high toxicity and resistance to environmental technology was specifical y developed for the extraction Solid Phase Extraction degradation (persistent organic pol utant), production and of polychlorinated biphenyls (PCBs) and related aromatic distribution of PCBs have been banned since the 1970's. compounds from transformer, waste and mineral oil. Because of their stability and persistence in the Under normal-phase conditions, PCB retention is facili- environment, PCBs are still monitored routinely and tated via interaction between the SPE phase's electrophilic heavily regulated. A common sample matrix encountered sulfur atom and the pi-electron cloud formed from in PCB analyses is oil used in dielectric, hydraulic, and heat aromatic rings inherent with PCBs.
transfer systems. There are numerous sample prep The phase is first conditioned with acetone to remove techniques currently available for PCB analysis in trans- residual moisture from the phase. This is a critical step. former oil ranging from sulfuric acid extraction (1) to SPE Any residual moisture on the phase wil negatively affect cleanup using silica gel, Florisil® (2), and/or Alumina (3). resolution and selectivity during extraction. The sulfoxide Most of these techniques are able to achieve lower limits phase is then equilibrated with hexane and a diluted oil of detection in the range of 5-10 ppm. However, as more sample (1:1 v/v with hexane) is loaded onto the packed transformers are decontaminated and waste sites undergo tube. Increasing volumes of hexane are then applied. treatment/remediation, lower limits of quantitation will be As the hexane wash solvent passes through the car- required to accurately determine PCB levels. This is a tridge, PCBs are preferential y retained/retarded on the chal enge because endogenous hydrocarbons found in SPE phase whereas endogenous sample interferences (e.
transformer oil behave similarly to PCBs during sample g., long chain hydrocarbons) are eluted from the phase preparation. As a result, they are often co-extracted with in the early fractions. Subsequent fractions are then PCBs and can interfere with subsequent GC-MS analyses eluted and col ected in later fractions for subsequent and possibly damage the GC instrument. GC-QMS or GC-HRMS analysis. Figure 1. Supelclean Sulfoxide SPE Separation of PCBs and Aliphatic Hydrocarbon
Figure 2. Supelclean Sulfoxide SPE Tube Interferences Prior to GC Analysis
Glass, 6 g/20 mL (55252-U) PCBs were extracted from oil and analyzed via GC-QMS using procedure described in Table 2. Figure 3 describes elution profile of PCBs vs. transformer oil (aliphatic hydrocarbons). As described in Figure 3, aliphatic hydro-carbons (oil interferences) are poorly retained on the Table 1. Supelclean Sulfoxide SPE Extraction Method sulfoxide SPE phase and elute off the packed bed within for PCBs in Transformer Oil the first 10-12 mL elution fraction. The retained chlorobi- Supelclean Sulfoxide SPE Tube, Glass 6 g/20 mL (55252-U)
phenyl congeners (CBs) are more strongly retained and 1. Condition the SPE phase with 20 mL acetone elute in the second 25 mL fraction. (removes residual moisture from the phase).
2. Equilibrate the SPE phase with 40 mL of hexane. Excellent Recovery and Lower Quantation
3. Load 0.4 mL diluted oil sample.
4. Elute aliphatic hydrocarbons (oil interferences) Insulation oil was spiked with PCBs at the total level of with 12 mL hexane 5. Elute PCBs with 25 mL hexane 3.7 mg/kg, extracted using Supelclean Sulfoxide SPE and 6. Col ect PCB fraction and concentrate under nitrogen analyzed via GC-QMS using the procedure described in for subsequent GC-QMS analysis (5) Table 2. Recovery was determined against 13C-label ed PCB internal standards. An average Recovery ± RSD of 98.5 ± Extraction and Analysis of PCBs in Transformer Oil
4.2 % was achieved for mono- to octa-chlorobiphenyls. 6 g of Supelclean Sulfoxide SPE was packed into a glass Concentrations of nona- to deca-chlorobiphenyls in the 20 mL SPE cartridge (17 mm I.D. x 137 mm L) (Figure 2). sample were lower than detection limits of the GC-QMS Solid Phase Extraction Commercial insulation oil (Japan Industrial Standard JIS system. Mono- and di-chlorobiphenyls in other samples C2320-1999, insulating oil, Class 1-2/4, paraffin oil) was having lower PCB concentrations were not determined spiked with a Kanechlor PCB mix at the total levels of 3.7 due to elution overlap with the tail-end of oil interfer-ences during sulfoxide SPE processing. This is of minor (US and Canada only) ppm (mg/kg) and diluted with hexane (1:1 v/v). The oil samples were extracted using the procedure described in concern because the primary PCB homologues of concern Table 1, and analyzed via GC-QMS using a 5% phenyl/ 95% in transformer oil samples consist of the tri- to heptachlo- methylpolysiloxane column and Agilent 5973N MSD (5). (continued on page 18) Figure 3. Elution Profile of Oil Interferences and PCB Congeners from Sulfoxide SPE / 814-359-3441 technical service: 800-359-3041 Elution Volume (mL, hexane) dering: 800-247-6628 or Table 2. Observed Concentrations of PCB Homologues of a PCB-fortified Insulation Oil Sample (n = 3).
tetra- penta- hexa- hepta- octa- nona- deca-
(continued from page 17) robiphenyls. Note that using the assay described in this + Featured Products
report, spike levels at the range of 0.045 - 0.9 mg/kg Description
(ppm) for the individual PCBs were able to be determined.
Supelclean Sulfoxide SPE
Glass SPE Tube, 6 g/20 mL (17 mm I.D. x 137 mm L), pk 5 Polypropylene SPE Tube, 3 g/6 mL, pk 30 In this report, we demonstrated the utility of a new silica-bonded sulfoxide SPE phase for the normal-phase extraction of PCBs (and possible related aromatic com- Related Products
Solid Phase Extraction pounds) from difficult sample matrices such as transform- Description
er oils. Because aliphatic hydrocarbons are often co- Empty Glass SPE Tube (17 mm I.D. x 137 mm L) with PE frit, extracted with PCBs using conventional SPE methods, 20 mL, with PE frit, luer cap, and screw-top cap, pk 5 Frit Insertion Tool for 20 mL Glass SPE tube lower limits of detection (< 5 ppm) are often difficult to Large Volume Reservoir (25 mL) for 6 mL SPE tubes, achieve. Sulfoxide SPE al ows for the user to separate Large Volume Reservoir (25 mL) for 6 mL SPE tubes, aliphatic hydrocarbon interferences from PCBs prior to GC SLB-5ms Capillary GC Columns
analysis using a generic/simple method. By removing this 15 m x 0.10 mm I.D., 0.10 µm key matrix interference prior to analysis, detection limits of 20 m x 0.18 mm I.D., 0.36 µm 30 m x 0.25 mm I.D., 0.25 µm less than 0.5 ppm are readily achieved. 30 m x 0.53 mm I.D., 0.50 µm 30 m x 0.53 mm I.D., 1.0 µm SPB-608 Capillary GC Columns
30 m x 0.25 mm I.D., 0.25 µm 1. Copland et al. Environ. Sci. Technol. 1982, 16, 121-124 30 m x 0.53 mm I.D., 0.50 µm 2. Solid Phase Extraction of PCBs from Transformer Oil and Waste Oil and Analysis By Equity-1701 Capillary GC Columns
Capil ary GC, Supelco Application Note 67,1998, T395067A 15 m x 0.10 mm I.D., 0.10 µm 3. Storr-Hansen et al. Chemosphere 1992, 24, 323-333 30 m x 0.25 mm I.D., 0.25 µm 4. Larsen et al. Chemosphere 1991, 23, 1077-1084 30 m x 0.53 mm I.D., 0.50 µm 30 m x 0.53 mm I.D., 1.0 µm 5. Numata et al. Anal. Chem. 2003, 75, 1450-1457 ! Related Information
For more information, please request the Supelclean Sulfoxide Data/Instruction Sheet, T707009, and Analysis of PCBs in Transformer Oil with a Sulfoxide Bonded SPE Phase and GC-MS, T408040, on the attached post card. These publications are available in electronic form only. Be sure to include your email address on the request form.
For a complete listing of our PCB standards and related reagents, please visit sigma-aldrich.com/pcb-standards DSD-DNPH Diffusive Sampler:
The Right Choice for Indoor Air Sampling of Carbonyls
Kristen L. Schultz
The DSD-DNPH is comprised of a porous polyethylene tube, which acts as the diffusive The DSD-DNPH diffusive sampler was first introduced in membrane, to which is attached a small Japan and was an integral device for monitoring carbonyls in polypropylene syringe used for the elution of indoor air, specifical y related to "sick building syndrome". the analytes from the adsorbent (1).
Sick building syndrome results from exposure to building Because the diffusive membrane is round, it materials that emit VOC's such as formaldehyde. Symptoms permits exposure from all sides, making it of formaldehyde sickness include coughing, burning eyes, unique compared to other diffusive samplers. nose bleeds, and sinus infections. Common building materi- Silica gel coated with 2,4-dinitrophenylhydra- als known to emit formaldehyde are: adhesives, paints, zine (DNPH) acts as the adsorbent and moves plywood, particle board, and wal paper. Under hot, humid from the diffusive end during sample col ection conditions, formaldehyde lets off toxic fumes which are to the syringe end for sample extraction, by especial y harmful to children with young lungs. The inverting the device. Aldehydes and ketones National Institute of Health Sciences (NIHS) in Japan conduct- diffuse through the membrane reacting with ed a study using the DSD-DNPH diffusive sampler from April DNPH to form stable derivatives. The DNPH- 2000 to March 2004. Now this unique device is available in derivatives are then eluted with acetonitrile and the US and offers the fol owing benefits: analyzed by high performance liquid chroma- tography (HPLC).
l Specified in OSHA 1007 Method for Determination of Aldehydes Comparison to Active Sampling
l Col ection and analysis of carbonyls without (US and Canada only) transfer of the adsorbent, which minimizes Sampling Rate: Paral el measurements were made with the risk of contamination the DSD-DNPH cartridges (28221-U) and active sampling l High-purity adsorbent provides col ection of ppb levels of a wide range of carbonyls cartridges using EPA Method IP-6A/TO-11, widely used as in a convenient, easy-to-use configuration a conventional method for active sampling of carbonyl l Excel ent uptake rates-faster, stable for compounds in environmental air. It is possible to obtain the wind, temperature and humidity sampling rate of the DSD-DNPH sampler from the compari- l Stable blank data – important for LOQ son with the known sampling rate of the active sampling. Active sampling was conducted using high precision l Versatile – use for indoor air, personal sampling, and ambient air apparatus (100 mL/min) composed of a mass flow control er (continued on page 20) Figure 1. Relationship Between Diffusive Sampling Method (DSD-DNPH) and Active Sampling Method on Formaldehyde, Acetaldehyde and 2-butanone (µg/m3); Sampling Period = 24 hours, N=188 / 814-359-3441 technical service: 800-359-3041 y = 0.99x – 0. Diffusive Sampling (DSD-DNPH) Diffusive Sampling (DSD-DNPH) Diffusive Sampling (DSD-DNPH) Relationship between 2-hour and 7-day monitoring data. Data collected using a 2–hour monitoring period are presented as mean values for 7 days and are plotted as ordinate. Seven-day monitoring data are plotted as abscissa.
dering: 800-247-6628 or (continued from page 19) Effect of Face Velocity
(model SEC-400 MARK3; STEC inc., Kyoto Japan), a wet gas The effect of air velocity on the sampling rate was studied meter (WS D-1A; Shinagawa Co., Tokyo), Supelco LpDNPH by moving the DSD-DNPH device in indoor air. Twenty DSD- S10L (505358) cartridges and Ozone scrubber (505285) to samplers were fixed at intervals of 10 cm on a 2 meter rod col ect carbonyl compounds. Samples were simultaneously (Figure 2), and then rotated by an electric motor at 48 rpm col ected in indoor and outdoor air for 24 hours. Col ected for 24 hours. A sampler, which was fixed 1 meter from the amounts were measured by HPLC.
central pivot of the rod corresponded to a face velocity of When one specific sampling rate of carbonyl compound 5.0 m/s. Wind velocity demonstrated little influence on the (e.g. formaldehyde) is determined from experimental data, DSD-DNPH method. The DSD-DNPH sampler indicated little the sampling rates of various other carbonyl compounds susceptibility to the face velocity because the rounded tube can be calculated from the ratio of diffusion coefficients, structure is omni-directional.
because the sampling rate is proportional to the diffusion The sampling amounts of formaldehyde and acetalde- coefficient. The diffusion coefficients can be obtained from hyde increased slightly depending on face velocity. RSD Ful er's equation or Graham's Law.
values for formaldehyde and acetaldehyde concentrations The values by diffusive sampling were in fair agreement were 5.5% and 8.6% respectively with a face velocity from with the values by active sampling. Figure 1 (pg 19) shows 0 to 5.0 m/s1.
the comparisons between the two methods concerning formaldehyde, acetaldehyde and 2-butanone at 188 data points. The coefficients of determination for formaldehyde, Is daily monitoring of indoor aldehydes necessary? The acetaldehyde and 2-butanone were 0.970, 0.961 and 0.971 DSD-DNPH demonstrates that it is sufficient to measure respectively. The slopes of the regression line were 0.95, formaldehyde every 7 days. Continuous sampling was performed for 24 hours and 7 days in 24 homes and results 0.96 and 0.99 respectively. It is thought that the sampling rate calculated from Graham's law is reasonable for the demonstrate that daily changes of formaldehyde during the DSD-DNPH method because good agreement was found measurement period for 7 days showed very large variation between the results obtained from diffusive and active and ranged from 16-170 µg/m3 (mean 86 µg/m3). Concen- sampling. Table 1 demonstrates these findings.
trations of formaldehyde estimated by the seven-day sampling method were nearly equal to the mean value The concentrations in Table 1 were mean values obtained calculated from the 24-hour sampling period measured from paral el measurements made with active sampling over 7 days. This confirmed that the concentration of compared to the diffusive sampling using the DSD-DNPH formaldehyde could be precisely monitored by 7 day device of compounds in indoor air of 188 houses through- continuous sampling. (2) out Japan from November 2001 to March 2002.
Table 1. Diffusion Coefficient (D), Sampling Rate (R), and Mean Concentration (C) of Carbonyl Compounds Calculated from Those Constant Values (n=188) ACTIVE SAMPLING
(cm2 S-1) (mL min-1) (µg m-3)
(cm2 S-1) (mL min-1) (µg m-3)
m,p-Tolualdehyde Figure 2. The Measurement of the Effect of Face Velocity + Featured Products
DSD-DNPH Diffusive Sampling Device + Related Products
Perforated Holder Female Luer Fitting to Tubing 5/32" Filtration Column w/o frit, 6 mL Plastic color-coded cap insert Visiprep™-DL vacuum manifold Figure 3. Relation Between Face Velocity and the Visi-1™ Sample Processor Amount Collected in the Sampler TO11/IP-6A Aldehyde/Ketone-DNPH Mix Formaldehyde-DNPH, 1 mL Acetaldehyde-DNPH, 1 mL Acetone-DNPH, 1 mL Acrolein-DNPH, 1 mL Propionaldehyde, 1 mL Discovery® RP-Amide, 25 cm x 4.6 mm I.D., 5µm ! Related Information
For more information, request the DSD-DNPH Product Flyer T408065 (KIX) and A High Efficiency Diffusive Sampler for the Determination of Aldehydes and Ketones in Ambient and Indoor (US and Canada only) Face Velocity (m/s1) Air, T400128 (DIC) on the attached postcard. These publications are available in electronic form only. Be sure to include your email address on the request form.
Since implementing the use of the DSD-DNPH diffusive sampler as one of the devices to effectively monitor indoor air quality in reference to carbonyl compounds in Japan; the Japanese government instituted regulation changes. These changes lowered acceptable levels of carbonyl compounds for residential housing. In addition, guidelines are provided for construction and use of related building materials (3).
1. S. Uchiyama and S Hasegawa, "A Reactive Sensitive Diffusion Sampler for the De- termination of Aldehydes and Ketones in Ambient Air", Atmospheric Environment, 1999, 33, 1999-2005. 2. S. Uchiyama, S Aoyagi, ad Ando, Masanori, "Evaluation of a Diffusive Sampler for Measurement of Carbonyl Compounds in Air", Atmospheric Environment, 2004, 38, 6319-6326.
/ 814-359-3441 technical service: 800-359-3041 3. Building Guidance Division, Housing Bureau: Ministry of Land, Infrastructure and Transport The amended Building Standard law on Sick House Issues. Japan, July 1,2003 "Instructions Regarding the Building Standard Law on Sick-House issues" "Overview of Countermeasures Regarding Sick House Issues under the Amended Building Standard Law" TRADEMARKS: Agilent - Agilent Technologies; Ascentis, CHIROBIOTIC, CHROMASOLV, CYCLOBOND, Discovery, Fluka, SP, Supelclean, Supelco, SupelMIP, Visi-1, Visiprep – Sigma-Al-
drich Biotechnology LP; AutoSystem, PerkinElmer - PerkinElmer Corp.; Carbowax - Union Carbide Chemicals & Plastics Technology Corp.; Florisil - US Silica Company; FocusLiner
- SGE International Pty Ltd.; Shimadzu - Shimadzu Corp.; Varian - Varian Associates Corp.; Waters - Waters Associates, Inc.
dering: 800-247-6628 or New! EPA Method 8270 LCS Mixes with Improved Stability
Steve Cecil, Jim Walbridge, Vicki Yearick
l Both LCS Spiking solutions are special y formulated to increase analyte stability including anilines and benzidines, while still providing Low spike recoveries for EPA Method 8270 are often for a water-soluble matrix.
seen for aniline and benzidine compounds in the LCS* l Our chemists have also addressed temperature, mixes. Sigma-Aldrich laboratory studies of these low light and oxygen stability issues.
recoveries have determined the cause to be interactions l Temperature has been determined in the of aniline and benzidines with other mix components Supelco laboratory studies to be the single largest contributor to shelf-life degradation. To ensure and/or the sample matrix. In addition to reactive the integrity of our spiking mixes, we ship them stability, our studies show low recoveries are also seen on dry ice. End-users should store the solutions in the freezer at -15 °C or colder, as noted on the for these compounds with increased exposure to oxygen, spike mixes documentation.
light and temperature.
l Reactivity with oxygen is avoided by blanketing Sigma-Aldrich chemists have designed two new the mixes with an inert gas when preparing and ampulizing the new mixes.
Supelco brand 8270 LCS Spike mix formulations to better l UV light degradation is minimized through the meet the spike-recovery requirements when performing use of non-UV emitting lights during production semi-volatile assays utilizing SW-846 methodologies. and the use of amber glass for storage.
These new 78-component LCS spiking standards are l Both spike mixes are offered in convenient 25 mL engineered for the improved stability need in today's volumes and include detailed lot specific mix preparation and analytical testing results.
EPA 8270 LCS Spike Mix 100 µg/mL each component in methanol:dichloromethane:benzene (90:9.4:0.6) EPA HC 8270 LCS Spike Mix 200 µg/mL each component in methanol:dichloromethane:benzene (80:18.75:1.25) Azobenzene Benzoic acid Dibutyl phthalate Benzyl alcohol Benzyl butyl phthalate Diethyl phthalate * LCS (Laboratory Control Sample/Blank Spike) Spikes are typical y required when sample matrix spike recoveries are determined to be outside the control limits. LCS standards are prepared by spiking known concentrations of target analytes into clean sample matrixes. The spiked LCS mix is then subjected to the same sample preparation and analysis protocols as the sample. LCS spike recoveries are calculated and used to determine the analytical accuracy of the method. Did you know.?
A safe and inexpensive method for removing the top from a 2 mL glass ampul is to use a Sigma-Aldrich ampul breaker (Z122904). Simply insert the top of the ampul into the breaker and snap off the top. The ampul top is retained in the breaker for safe disposal.
Prescreened, In-Stock Chemicals:
B100 Biodiesel, Epigallocatechin, Butyl Mercaptan, Tetrabutyltin and More
What do these compounds have in common? They are exam- ples of the thousands of prescreened, in stock chemicals available through the Sigma-Aldrich custom standards group. We can formulate, test, and package custom standard solutions to meet your needs for all your chromatographic applications. Our custom standard chemists will gladly discuss stability and solubility concerns with you, and make suggestions where needed to improve the quality of your purchase.
You can rely on Sigma-Aldrich custom standard solutions to include: l raw materials and solvents screened for identity and purity l your choice of gravimetric, qualitative, and quantitative testing l packaging choices from ampuls to bottles l manufacturing processes fol owing the guidelines of ISO 9001/2000 l proper handling of light- and/or oxygen-sensitive chemicals l documentation and Material Safety Data Sheets l free technical support l strict adherence to all shipping regulations ! Related Information
If you are interested in a customized standard. please email us at [email protected], or complete the on-line custom standards (US and Canada only) quote request form, available 24 hours a day – 7 days a week, at our website sigma-aldrich.com/standards Are Your Vials & Inserts Compatible?
Table 1. Differing Specifications for US & European Vials & Inserts The I.D. of 2 mL vials can vary by as much as US Manufacturer
0.3 mm. As a result of this variation, your vial insert may not properly fit, costing you time, money and causing frustration.
Standard I.D. (mm) 5.04–5.06 4.58-4.68 5.18-5.22 4.95-5.00 Due to a lack of worldwide standards, this 5.94–6.08 5.67-5.77 6.18–6.21 5.98-6.00 problem is magnified when comparing products manufactured in the US and Europe. Compatible Vials & Inserts from US and European Manufacturers These differences are shown in Table 1.
/ 814-359-3441 technical service: 800-359-3041 To ensure these parts fit together properly, US Crimp Neck Vial, Large Opening, 2 mL, 12 x 32 mL
we recommend that your vials and inserts be purchased from the same manufacturer.
US Glass inserts
Sigma-Aldrich offers a variety of compatible 0.2 mL, 6 mm x 29 mm with bottom spring vial and insert products. For help with product European Crimp Neck Vial, Large Opening, 1.5 mL, 11.6 x 32 mm
selection, email Sigma-Aldrich Technical Service at [email protected] or visit our website European Glass inserts
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