Thermo Scientific AQUASIL C18 HPLC Columns

1 Exceptional Chromatography of Polar CompoundsPart of Thermo Fisher ScientificThermo Scientific AQUASIL C18 hplc Columns Technical Guide Version 2 IntroductionThe Thermo Scientific AQUASIL C18 columnsprovide a versatile C18 phase for a widerange of a C18 phase with a hydrophilicend-capping, AQUASIL C18 Columns offer aunique material for reversed phase chroma-tography, offering alternative selectivity, upto twice the retention for polar compounds,and no phase collapse under 100% aqueousconditions. Ideal for use with LC/MS, thesecolumns maintain selectivity with reducedconcentrations of buffers and this Technical Guide, we review theAQUASIL C18 packing, designed to gobeyond the limitations of traditional C18packing materials, including: Different retention and selectivity toconventional C18 Excellent peak shapes for basic,acidic and neutral compounds Polar molecule retention twice asstrong as conventional C18 Compatible with 100% aqueous mobile phase Excellent results with low buffer concentrations Stable for LC/MS applicationsChromatographic CharacterizationPackings that offer additional interactionmodes give rise to quite different retentionbehavior and selectivity.

2 Additionally, analyteswith the greatest polar functionality willtypically show significant changes in selectivity and 1 illustrates the behavior differ-ences between the AQUASIL C18 column andthe Thermo Scientific BetaBasic 18 column(a C18 column that is highly base deactivatedand densely bonded and also has a verysimilar percent carbon value).Figure 1a demonstrates that the AQUASILC18 column is slightly less retentive thanthe BetaBasic 18 column where analyteinteractions are based purely on hydrophobic(or dispersive) interactions. The AQUASIL C18packing was designed for the reversed phaseseparation of polar molecules. Despite itsrelatively high concentration of C18 groups,it also has hydrophilic sites that help to provide increased retention of highly polarwater soluble compounds (Figure 1b, 1c). Thermo Scientific AQUASIL C18 hplc Columns212neucom345 Figure 1: Chromatographic Retention Behavior AQUASIL C18 vs BetaBasic 18 Columns67812345678 BetaBasic 18 AQUASIL C18a) Less retentive for neutral compoundsb) More retentive for polar basic compounds123456123456 Sample1.

3 Uracil2. Benzene3. Ethylbenzene4. Propylbenzene5. Butylbenzene6. Pentylbenzene7. Hexylbenzene8. HeptylbenzeneColumns:5 m, 150 x mmEluent:25% H2O / 75% mL/minDetector:UV @ n-Acetylprocainamide4. Caffeine5. n-Propionylprocainamide6. PhenolColumns:5 m, 150 x mmEluent:90% 50 mM KH2PO4,pH / 10% mL/minDetector:UV@ 254 ABB-001 BetaBasic 18 AQUASIL C18c) More retentive for polar acidic compounds12341234 Sample1. Uracil2. Phloroglucinol3. Resorcinol4. PhenolColumns:5 m, 150 x mmEluent:80% FormicAcid/20% mL/minDetector:UV @ 254 polcomBetaBasic 18 AQUASIL C1802 4 6810 MinThermo Scientific AQUASIL C18 hplc ColumnsFigure 1b shows how the AQUASIL C18column offers the nearly twice the retentionof several polar, basic compounds whencompared to a BetaBasic 18 column. Theretention of basic compounds and polar acidiccompounds on the AQUASIL C18 column areboth significantly increased compared to theBetaBasic 18 column. This illustrates howuseful the AQUASIL C18 column can bewhen increased retention of polar compoundsor alternative selectivity is Retention ofPolar CompoundsPolar compounds often elute near or at theunretained marker when run on typical C18 hplc Columns .

4 AQUASIL C18 Columns provideadditional analyte-ligand interactions toreversed phase hydrophobic interactions,leading to increased retention of analytes withpolar functionality. AQUASIL C18 columnsmaintain retention of neutral compoundswhile offering increased retention for bothacidic and basic compounds. Applications Highly Polar Compounds Nucleosides and Nucleotides Organic acids Vitamins Peptides CatecholaminesChromatographic InteractionsDispersive interactions are the primaryinteractions generally associated withretention using traditional alkyl C18 type packings. Secondary interactions associated withresidual silanols have been significantlyreduced by end-capping, improvements insilica quality and increased density of thederivatized ligand. Therefore, silanol inter-actions that previously gave rise to broadtailing peaks for basic analytes have beensomewhat eliminated. These secondaryinteractions are also responsible in part forthe retention of compounds with polar functionality, either by hydrogen bondinginteractions or via ion exchange progressive elimination of the secondary silanol interactions has resultedin Columns that give good peak shape forbasic compounds but reduced retention ofpolar compounds in general.

5 AQUASIL C18columns provide an excellent combinationof traditional reversed phase interactionsand polar interactions to retain morepolar SIZE PORE SIZE CARBON LOAD SILICA TYPEAQUASIL C183 and 5 m100 12%High purity, base deactivatedAQUASIL media combines C18 with hydrophilicendcapping AQUASIL Phase Specifications3 This is illustrated in Figure 2 whichdemonstrates the capability of the AQUASILC18 column s ability to separate catechol-amines without ion pair reagents in a highlyaqueous mobile phase. Highly Aqueous Mobile PhasesThe inclusion of polar functionality to thestationary phase also increases the wettingcharacteristics of the packing in highlyaqueous mobile phases. The AQUASIL C18 column can be run in 100% aqueous mobile phase conditions(Figures 3 and 4) and shows no tendencytowards phase collapse. Phase collapse isoften seen with C18 packings unless a smallamount of organic solvent (1-5%) is addedto the mobile phase. As a result of phase collapse, theretention and selectivity of the phase arelost and the column must be regeneratedusing a pure organic solvent wash.

6 TheAQUASIL C18 packing is immune to thisfolding due its unique polar 4 demonstrates that reproduciblechromatography is maintained even afterflushing with 100% aqueous mobile phasefor 113 :1. Epinephrine2. L-DOPA3. DL-Tyrosine4. Vanillylmandelic acidAQUASIL C18, 5 m, 150 x mmEluent:98% KH2PO4, pH H3PO4/ 2% mL/minDetector:UV @ 210 Figure 2: Catecholamines123402468101214 Min775-0461. 5'-CTP 5'-CDP 5'-CMP 5'-GDP 5'-GMP 5'-ATP 5'-ADP 5'-AMP C18, 5 m, 150 x KH2PO4, pH with mL/minDetector:UV @ 260 Figure 3: Separation of nucleotides with 100%aqueous mobile pahse12345775-008 Sample:1. Tartaric acid2. Malonic acid3. Fumaric acid4. Succinic acidAQUASIL C18, 5 m, 150 x KH2PO4+ H3PO4, pH mL/minDetector:UV @ 210 Figure 4: AQUASIL C18 compatibility with 100%aqueous mobile phase1234687432102 4 6 Min775-009 Initial RunAfter 113 Hoursof Flushing withMobile Phase4 Thermo Scientific AQUASIL C18 hplc ColumnsReduced Buffer Concentrationsand Increased MS SensitivityAt any concentration, additives such as trifluoroacetic acid can cause ion suppressionand consequently reduce sensitivity inLC/MS methods.

7 The choice of hplc columnis of key importance for LC/MS applications,since the properties of the bonded stationaryphase and underlying silica can stronglyinfluence the concentration of enhance ionization, it is good practiceto use volatile mobile phase additives inLC/MS methods. The addition of acidicmodifiers such as formic acid or TFA is commonplace when analyzing proteins andpeptides by reversed phase additive solvates the analyte, displacingwater molecules and creating a morehydrophobic analyte with stronger retentionon traditional C18 packings. The AQUASIL C18 packing can retainpeptides in their water soluble state, reducingthe need for TFA as an additive. The examples shown in Figure 5 illustrate howthe AQUASIL C18 column can be used atvery low TFA concentrations while maintainingretention and performance of many of thepeptides of interest. Low level additive con-centrations (Figure 5c) also offer the rewardof increased sensitivity for MS. This is animportant consideration when trying toidentify trace quantities of a drug compoundor impurity that may normally disappear intothe noise of the baseline of the MS 5: Tryptic Digest of -Lactoglobulina)b)c)504 8 1216 Min775-007 Sample:1.

8 Uracil2. Lidocaine3. Chlorpheniramine4. Acid5. DoxepinAQUASIL C18, 5 m, 150 x mmEluent:A: KH2PO4+ H3PO4, pH : ACN 75% A / 25% mL/minDetector:UV @ 254 Acid Base Mix1234502468 Min775-045 Sample:1. Vitamin C2. Thiamine (Vitamin B1)3. Pyridoxine (Vitamin B6)4. Niacinamide (Vitamin B3)5. Vitamin B56. Folic Acid7. d-Biotin (Vit. H)8. Vitamin B29. Vitamin B12 AQUASIL C18, 5 m, 50 x mmEluent:A: K2 HPO4, pH : 50% ACN / 50% , pH 4% B to40% B in 9 minDetector:UV @ 205 Water-Soluble Vitamins12345051015 Min775-037bSample: A)1. Lutein2. Zeaxanthin3. & 4. Lycopene5. -cryptoxanthin6. -Carotene7. -CaroteneSample:B)1. Retinol2. Tocol3. -Tocopherol4. -Tocopherol5. -TocopherolAQUASIL C18, 5 m, 250 x mmEluent:83%ACN / 16%Dioxane/ 1% mL/minDetector: chromatogram A: UV@450 nmchromatogram B: UV@325/300 nmFat-Soluble Vitamins0510 Min775-200 Sample:Alberta Peptide Mix(heptapeptides) AQUASIL C18, 3 m, 100 x mmEluent:A: TFA in H2O B: TFA in ACN0%B to 50%B in 20 mL/minDetector:UV @ 210 Peptides1234504812 Min775-041 Sample:1.

9 Benzoic acid2. 4-Fluoro benzoic acid3. 2-Toluic acid4. 4-Toluic acid5. 2,4,6-Trimethyl benzoic acid6. 2,5-Dimethyl benzoic acid7. 2,4-Dimethyl benzoic acidAQUASIL C18, 5 m, 150 x mmEluent:50% MeOH / 50% KH2PO4, pH mL/minDetector:UV @ 230 Temperature: +/- CBio-Remediation Acids12345048 12 Min775-038 Sample:1. Lincomycin2. Internal Standard3. Clindamycin-b4. 7-epiclindamycin5. ClindamycinAQUASIL C18, 5 m, 250 x mmEluent:A: MeOHB: 4g/L dl-10- camphorsulfonicacid, Adjust to pH 6 with HCL or NaOH A / 40% mL/minDetector:RIClindamycin1234567892B3 B4B5B1B1A2A3A4A5A6A7A67 Data courtesy ofJames D. Stuart andReena M. Joseph,Department ofChemistry, Univ ofConnecticut, Storrs, CT 6 Example ApplicationsThermo Scientific AQUASIL C18 hplc ColumnsProcainamides Comparison04812 Min775-042 Sample1. Benzoic acid2. 2-Toluic acid3. 4-Toluic acid4. 2,4,6-Trimethylbenzoic acid5. 2,4 & 2,5-DimethylbenzoicacidAQUASIL C18, 5 m, 150 x mmEluent:50% MeOH/50% KH2PO4, pH mL/minDetection:UV @ 230 Temperature: 22 CGround Water ExtractSample:1.

10 Uracil2. Procainamide3. n-acetylprocainamide4. Caffeine5. n-propionylprocainamideBetaBasic 18, 5 m, 150 x mmEluent:10% ACN/90% KH2PO4, pH mL/minDetection:UV @ 254 AQUASIL C18, 5 m, 150 x mmEluent:10% ACN / 90% , pH mL/minDetection:UV @ 254715-036715-01602468 Min02 4 6 8 10121416 Min123451234512345 Organic Acids in Highly Aqueous Mobile Phase 0246 8 Min775-003 Sample:1. Cytidine2. Uridine3. Adenosine4. Guanosine5. ThymidineAQUASIL C18, 5 m, 150 x mmEluent:A: KH2PO4, & , pH : H2OC: ACN B/5% mL/minDetection:UV @ 260 NucleosidesSample:1. Oxalic Acid2. Tartaric Acid3. Pyruvic Acid4. Malic Acid5. Lactic Acid6. Acetic Acid7. Acid8. Maleic Acid9. Succinic Acid10. Fumaric Acid11. Propionic Acid12. Butyric AcidAQUASIL C18, 5 m, 250 x mmEluent:1% ACN / 99% KH2PO4, pH mL/minDetection:UV @ 210775-09202 4 6 8 10121416 1820 22 Min123451234587611109127 AQUASIL C18 Columns are available in other column formats. Please contact your local customer support for more InformationAQUASIL C18 ColumnsParticle SizeLength (mm) m m m m m ID3 m3077503-03463077503-03403077503-0330307 7503-03213077503-0310305077503-054630775 03-05403077503-05303077503-05213077503-0 5103010077503-10463077503-10403077503-10 303077503-10213077503-10103015077503-154 63077503-15403077503-15303077503-1521307 7503-1510305 m3077505-03463077505-03403077505-0330307 7505-03213077505-0310305077505-054630775 05-05403077505-05303077505-05213077505-0 5103010077505-10463077505-10403077505-10 303077505-10213077505-10103012577505-124 63077505-12403077505-12303077505-1221307 7505-12103015077505-15463077505-15403077 505-15303077505-15213077505-151030250775 05-25463077505-25403077505-25303077505-2 5213077505-251030 Other column dimensions are also available.