atlantis™ columns applications notebookquimica.udea.edu.co/~carlopez/cromatohplc/wa20720.pdf ·...

Applications Notebook

©2002 Waters Corporation. Waters and Atlantis are trademarks of Waters Corporation 1

Enhanced Retention of Polar Compounds with Atlantis™ as Comparedto Embedded Polar Group Containing Stationary Phase

Comparison of the retention time of polar analytes on a packing with embedded polargroup (bottom) to the Atlantis™ dC18 packing (top). Mobile phase: 10 mM ammoniumformate, pH 3.0. Flow rate: 1.2 mL/min. Sample: 1. Thiourea, 2. 5-Fluorocytosine,

3. Adenine, 4. Guanosine-5’-monophosphate, 5. Thymine.

An Ideal Column

As can be seen by the key attributes of the Atlantis dC18 columns, Watershas succeeded in producing an ideal column for reversed-phase HPLC.Atlantis™ columns have succeeded where embedded polar groupcolumns have fallen short: 100% aqueous mobile phase compatibilityand enhanced retention for polar compounds. In addition, the difunc-tional bonding chemistry of the Atlantis™ packing material providesexcellent stability under acidic mobile phase conditions. The packingdoes not exhibit excessive retention for hydrophobic compounds, andavoids the tailing and extreme retention for bases observed with unend-capped stationary phases. It is also compatible with all detectors, includ-ing the highly sensitive MS detector. Of course, during the developmentof the Atlantis™ columns packing material, Waters also paid close atten-tion to its reproducibility. As with any one of the modern Waters pack-ings, the batch-to-batch reproducibility is incontestable.

Key Attributes of Atlantis™ Columns

• Superior retention of polar compounds• Operates and thrives in 100% aqueous mobile phases• Excellent peak shape with Mass Spectrometry compatible mobile phases• Difunctional bonding chemistry results in:

- Extended column lifetime at low pH- Enhanced low pH stability

• LC/MS compatible with ultra-low column bleed• Enhanced polar compound retention

without infinite non-polar compound retention

For More Information

To learn more about Atlantis™ columns pleasevisit the Waters website at www.waters.com.

Polar compounds are extremely difficult to retain and separate usingreversed-phase chromatography. One of the reasons is that columnmanufacturers have focused on producing stationary phases that provide enhanced retention for amine-containing bases. In order to retain bases while still maintaining good peak shape, these phasestend to have high ligand densities and small pore sizes. However, in the creation of such phases, retention of polar compounds is often sacrificed. This is due to the column’s inability to function in the aqueous or highly aqueous mobile phases that are necessary to retainthese hydrophilic compounds. Waters studied the effects of the stationary phase on polar retention and created a column that retainspolar compounds, provides excellent peak shape, and can operate inhighly aqueous mobile phases.

Atlantis™ Columns — An Intelligent Design

Optimizing pore size, ligand type, ligand density and endcapping results in the bestpossible peak shape, stability, dewetting and polar retention.

Embedded Polar Groups For Retention of HighlyPolar Compounds – Fact or Fiction?

Stationary phases that contain embedded polar groups (e.g., urea,amide, carbamate, ether, etc.) were designed for improved peakshapes for bases and to provide complementary selectivity ascompared to straight-chain alkyl phases. Another benefit of embeddedpolar group containing stationary phases is their compatibility withaqueous mobile phases. The embedded polar group holds onto thewater and increases the water layer at the surface of the pores. Thisaqueous compatibility is often confused with enhanced polar retentionsince the mobile phases necessary for polar compound retention areoften highly aqueous. Contrary to popular belief, stationary phasesthat contain embedded polar groups actually result in less retention forvery polar compounds. This is due to the shorter chain length of near-ly all phases that contain embedded polar groups. Another reason forthe shorter retention is the increased water content of the stationaryphase layer which yields an environment with a higher dielectric con-stant, and hence a weaker ionic interaction between cations (e.g.,amines) and the dissociated silanols on the silica surface.

Atlantis™ dC18 columns do not contain nor require an embedded polargroup. As described above, by carefully designing and understand-ing the stationary phase chemistry, aqueous compatibility and polarretention can be achieved using a straight-chain alkyl C18 phase. Anexample of the enhanced retention of Atlantis™ columns as comparedto a column that contains a stationary phase with an embedded polargroup is given in the following figure.

Introduction

Li d

12

3

4 5

Endcapping

LigandDensity

PoreSize

LigandType

Peak ShapeStability

DewettingRetention

Atlantis™ Columns Brochure

Literature Code 720000472EN

Atlantis™ dC18

Embedded Polar Group Column

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00Minutes

1 23

45

1

42

3

5

Table of Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Compound Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

List of Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Troubleshooting and Problem Solving Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-47

Care and Use Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48-55

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56

Compound Index2’-Deoxycytidine. . . . . . . . . . . . . . . . . . . . . . . 302’-Deoxyguanosine . . . . . . . . . . . . . 30,32,33,342’-Dexoyadenosine . . . . . . . . . . . . . . . . . . . . 302,4,5-Trihydroxybutyrophenone (THBP) . . . . . . . 242,6-Di-tert-butyl-4-hydroxymethylphenol . . . . . . . . 242-Acetamidophenol . . . . . . . . . . . . . . . . . . . . . 92-Amino-7-chloro-5-oxo-5H-[1]benzopyrano -[2,3-b]pyridine-3-carbonitrile . . . . . . . 39,40,41,422-Bromofluorene . . . . . . . . . . . . . . . 39,40,41,423,4-Dihydroxyphenylacetic acid (DOPAC) . . . 17,183,4-Dihydroxyphenylalanine (DOPA) . . . . . . 17,183-Aminofluoranthene . . . . . . . . . . . . 39,40,41,423-Methoxy-4-hydroxyphenylglycol (MHPG) . . . . . 184-Hydroxy-3-methoxymandelic acid (VMA) . . 17,184-Hydroxy-3-methoxyphenylacetic acid (HVA) . . 17,184-Hydroxybenzoic acid . . . . . . . . . . . . . . . . . 125-Fluorocytosine . . . . . . . . . . . . . . . . . . . . 14,295-Fluorouracil . . . . . . . . . . . . . . . . . . . . . . . . 295-Hydroxy-3-indoleacetic acid (5-HIAA) . . . . 17,186α-Methyl-17α-hydroxyprogesterone . 39,40,41,428-Bromoguanosine . . . . . . . . . . . . . . . . . . 39,408-Hydroxy-2’-deoxyguanosine . . . . . . . . 32,33,34A6677 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Acephate . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Acetaminophen . . . . . . . . . . . . . . . . . . . . . 9,19Acetanilide . . . . . . . . . . . . . . . . . 9,39,40,41,42Acetic acid . . . . . . . . . . . . . . . . . . . . . . . . . . 31Acetylsalycylic acid . . . . . . . . . . . . . . . . . . . . . 9Acyclovir . . . . . . . . . . . . . . . . . . . . . . . . . . . 5,6Adenine . . . . . . . . . . . . . . . . . . . . . . . . . 14,29Adenosine . . . . . . . . . . . . . . . . . . . . . . . . . . 30Amoxicillin . . . . . . . . . . . . . . . . . . . . . . . . . . 7,8Angiotensin I . . . . . . . . . . . . . . . . . . . . . . . . . 36Angiotensin II . . . . . . . . . . . . . . . . . . . . . . . . 36Arbutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Atenolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Benzocaine . . . . . . . . . . . . . . . . . . . . . . . . . 10Bradykinin . . . . . . . . . . . . . . . . . . . . . . . . . . 36Butacaine . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Butylated hydroxyanisole (BHA) . . . . . . . . . . . . 24Butylated hydroxytoluene (BHT) . . . . . . . . . . . . 24Caffeine . . . . . . . . . . . . . . . . . . 9,16,22,44,47Catechin . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Catechin gallate. . . . . . . . . . . . . . . . . . . . . . . 16Chlorpheniramine . . . . . . . . . . . . . . . . . . . 19,22Chlorthalidone . . . . . . . . . . . . . . . . . . . . . . . 21Cinoxacin . . . . . . . . . . . . . . . . . . . 25,26,27,28Cytidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Cytosine . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Dextromethorphan . . . . . . . . . . . . . . . . . . . . . . . 19Dibutyl phthalate . . . . . . . . . . . . . . . . . . . . . . 38Dimethyl phthalate . . . . . . . . . . . . . . . . . . . . . 38Dioctyl phthalate . . . . . . . . . . . . . . . . . . . . . . 38Diphenhydramine . . . . . . . . . . . . . . . . . . . . . 19Dopamine (DA) . . . . . . . . . . . . . . . . . . . . . . . 18Epicatechin . . . . . . . . . . . . . . . . . . . . . . . . . 16Epicatechin gallate . . . . . . . . . . . . . . . . . . . . . 16Epigallocatechin . . . . . . . . . . . . . . . . . . . . . . 16Epigallocatechin gallate. . . . . . . . . . . . . . . . . . 16Epinephrine (E) . . . . . . . . . . . . . . . . . . . . . 17,18Folic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Fumaric acid . . . . . . . . . . . . . . . . . . . . . . 12,31Furosemide . . . . . . . . . . . . . . . . . . . . . . . . . . 21Gallic acid . . . . . . . . . . . . . . . . . . . . . . . 12,16Gallocatechin gallate . . . . . . . . . . . . . . . . . . . 16Gentisic acid . . . . . . . . . . . . . . . . . . . . . . . . 12Ginsenoside Rb2 . . . . . . . . . . . . . . . . . . . . . . 23Ginsenoside Rc . . . . . . . . . . . . . . . . . . . . . . . 23Ginsenoside Rd . . . . . . . . . . . . . . . . . . . . . . . 23Ginsenoside Rg1 . . . . . . . . . . . . . . . . . . . . . . 23Guanine . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Guanosine . . . . . . . . . . . . . . . . . . . . . . . 18,24Guiacol glyceryl ether . . . . . . . . . . . . . . . . . . 19Guanosine-5-monophosphate . . . . . . . . . . . . . . 14Hydrochlorothiazide . . . . . . . . . . . . . . . . . . . . 21Hydrocortisone . . . . . . . . . . . . . . . . 39,40,41,42Insulin B Chain . . . . . . . . . . . . . . . . . . . . . . . 36Isophthalic acid . . . . . . . . . . . . . . . . . . . . . . . 37L-ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . 47L2275 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Labetolol . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Lactic acid . . . . . . . . . . . . . . . . . . . . . . . . . . 31Lauryl Gallate (LG) . . . . . . . . . . . . . . . . . . . . . 24Lidocaine . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Maleic acid . . . . . . . . . . . . . . . . . . . . . . 22,31Malic acid . . . . . . . . . . . . . . . . . . . . . . . . . . 31Melittin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Methamidophos . . . . . . . . . . . . . . . . . . . . . . . 43Metoprolol . . . . . . . . . . . . . . . . . . . . . . . . . . 15Nalidixic acid . . . . . . . . . . . . . . . . 25,26,27,28Naphtho(2,3-a)pyrene . . . . . . . . . . . 39,40,41,42Nicotinic acid . . . . . . . . . . . . . . . . . . . . . . . . 47Norepinephrine (NE) . . . . . . . . . . . . . . . . . . . 18Octyl Gallate (OG) . . . . . . . . . . . . . . . . . . . . 24Oxalic acid . . . . . . . . . . . . . . . . . . . . . . . . . 31Oxolinic acid . . . . . . . . . . . . . . . . . 25,26,27,28

Oxprenolol . . . . . . . . . . . . . . . . . . . . . . . . . . 35Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Perylene . . . . . . . . . . . . . . . . . . . . 39,40,41,42Phenacetin . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . 13Phenylephrine . . . . . . . . . . . . . . . . . . . . . . . . 22Phenylpropanolamine . . . . . . . . . . . . . . . . . . . 22Phthalic acid . . . . . . . . . . . . . . . . . . . . . . . . . 37Pindolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Procaine . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Propranolol . . . . . . . . . . . . . . . . . . . . . . . . . . 15Propyl Gallate (PG) . . . . . . . . . . . . . . . . . . . . 24Protocatechuic acid . . . . . . . . . . . . . . . . . . . . 12Pseudoephedrine . . . . . . . . . . . . . . . . . . . . . . 19Pyridoxal . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . 47Pyruvic acid . . . . . . . . . . . . . . . . . . . . . . . . . 31Renin Substrate . . . . . . . . . . . . . . . . . . . . . . . 36Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Serotonin (5-HT) . . . . . . . . . . . . . . . . . . . . 17,18Substance P . . . . . . . . . . . . . . . . . . . . . . . . . 36Succinic acid . . . . . . . . . . . . . . . . . . . . . . . . 31Sulfadiazine . . . . . . . . . . . . . . . . . . . . . . . . . 45Sulfamerazine . . . . . . . . . . . . . . . . . . . . . . . . 45Sulfamethazine . . . . . . . . . . . . . . . . . . . . . . . 45Sulfamethoxazole . . . . . . . . . . . . . . . . . . . . . 45Sulfathiazole . . . . . . . . . . . . . . . . . . . . . . . . . 45Syringic acid . . . . . . . . . . . . . . . . . . . . . . . . 12Tartaric acid . . . . . . . . . . . . . . . . . . . . . . . . . 32Terphthalic acid . . . . . . . . . . . . . . . . . . . . . . . 37Tert-butylhydroquinone (TBHQ) . . . . . . . . . . . . . 24Tetracaine . . . . . . . . . . . . . . . . . . . . . . . . . . 10Theobromine . . . . . . . . . . . . . . . . . . . . . . . . . 44Theophylline . . . . . . . . . . . . . . . . . . . . . . . . . 44Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Thiourea . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Thymidine . . . . . . . . . . . . . . . . . . . . . . . . . . 30Thymine . . . . . . . . . . . . . . . . . . . . . . . . . 14,29Triamcinolone . . . . . . . . . . . . . . . . 39,40,41,42Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . 13Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Uracil . . . . . . . . . . . . . . . . . . . . . . . . 29,41,42Uridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Vancomycin . . . . . . . . . . . . . . . . . . . . . . . . . . 46Vanillic acid . . . . . . . . . . . . . . . . . . . . . . . . . 12

2 ©2002 Waters Corporation. Waters and Atlantis are trademarks of Waters Corporation

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List of Applications

Acyclovir – Isocratic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Acyclovir – Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Amoxicillin – Isocratic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Amoxicillin – Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Analgesics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Anesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Arbutin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Aromatic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Aromatic Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Atlantis™ 100% Aqueous QC Batch Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

ß-Blockers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Catechins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Catecholamines – Isocratic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Catecholamines – Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Cold Medicine Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Cytochrome C Tryptic Digests (LC/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Diuretics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Drugs for Rhinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Ginsenosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Lipid Soluble Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Nalidixic Acid Antibiotics (UV - Isocratic) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Nalidixic Acid Antibiotics (UV – Gradient) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Nalidixic Acid Antibiotics (LC/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27-28

Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Organic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Oxidative Stress Markers – ACN, pH 3.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Oxidative Stress Markers – MeOH, pH 5.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33-34

Paclitaxel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Phthalic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Phthalic Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Polar and Non-Polar Compounds Test Mix – 2.1x30 mm column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39-40

Polar and Non-Polar Compounds Test Mix – 4.6x150 mm column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41-42

Polar Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Purine Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Sulfonamides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Vancomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Water Soluble Vitamins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47


Click on list, Type desired page number, Click OK

Little or no retention ofpolar compounds

Method requires 100% aqueous mobile phase for desired separation

Sudden loss of analyte retentionobserved when using highly aqueousmobile phase

Short column lifetime in acidicmobile phases

Retaining polar compounds on aconventional C18 column results in increasedor infinite retention of non-polar compounds

Severe peak tailing for polar basesis observed

Column bleed is observed on MS

Column to column reproducibility is inconsistent(e.g., selectivity, retention, etc.)

• Re-run samples using separate methodsfor polar compounds

• Increased method developmenttime and labor

• Loss of retention is observed

• Run organic modifier through column torewet and regenerate column

• Increased labor and solvent costs• Decreased throughput• Reproducibility issues

• High cost due to frequentcolumn replacement

• Increased instrument downtime• Retention time reproducibility issues

• Multiple columns are required to separateanalytes with a wide range of polarities

• Increased method development time,labor and column costs

• Decreased throughput

• Method fails system suitability guidelinesfor peak tailing

• Increased method development time

• Frequent cleaning of MS source• Incorrect or inconsistent results

• Increased labor costs due to individualcolumn QC testing

• Revalidate/redevelop method with eachnew batch of columns

• Polar compounds are retained longer withAtlantis™ columns

• One Atlantis™ column and method can be used forpolar and non-polar compounds

• Atlantis™ packing material is tested with highly polaranalytes in 100% aqueous conditions, ensuringits utility in aqueous conditions

• Atlantis™ columns don't lose retention in 100%aqueous mobile phases

• Less time spent rewetting columns resulting in lowerlabor costs

• Increased throughput

• The proprietary difunctional bonding chemistry ofAtlantis™ columns results in improved stability at lowpH and longer column lifetime

• Decreased costs associated with column replacementand instrument maintenance

• One Atlantis™ column and method can be used forpolar and non-polar compounds

• Easier and faster method development• Increased throughput

• Atlantis™ columns are optimally endcapped and provideexcellent peak shapes using MS compatible mobile phases

• Easier and faster method development

• Atlantis™ columns do not exhibit MS-detectablecolumn bleed

• Decreased instrument downtime and maintenance costs

• The stringent Atlantis™ packing material QC batch testseparates highly polar analytes in 100% aqueous mobilephase conditions

• Decreased method revalidation and development time

Problem Impact Solution and Benefit

Troubleshooting and Problem Solving Using Atlantis™ Columns


Acyclovir - Isocratic

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

USP Tailing = 1.34

V0 = 1.0

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µm Part Number: 186001329Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Composition: 87% A; 3% B; 10% CInjection Volume: 5 µLSample Concentration: 10 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795,2996 PDA

Acyclovir


USP Tailing = 1.00

V0= 1.0

Acyclovir - Gradient

USP Tailing = 1.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

V0 = 1.0

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µm Part Number: 186001329Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 90 0 105.0 85 5 10

Injection Volume: 5 µLSample concentration: 10 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA Acyclovir


Amoxicillin - Isocratic

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

V0 = 1.0 USP Tailing = 1.19

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µmPart Number: 186001329Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0Flow rate: 1.0 mL/minIsocratic Mobile Phase Composition: 87% A; 3% B; 10% CInjection Volume: 10 µLSample concentration: 40 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA

Amoxicillin



Amoxicillin - Gradient

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

V0 = 1.0

USP Tailing = 1.01

Conditions


(min) %A %B %C0.0 90 0 105.0 85 5 10

Injection Volume: 10 µLSample concentration: 40 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA

Amoxicillin

Analgesics

12

3

4

5

6

V0 = 1.83

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 1% HCOOH, pH 2.3Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 75 15 1010.0 30 60 10

Injection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 260 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing1. Acetaminophen 1.002. Caffeine 0.993. 2-Acetamidophenol 0.984. Acetanilide 1.005. Acetylsalicyic acid 0.966. Phenacetin 0.96

Acetaminophen Phenacetin

HN

O

HO

HN

O

O O

OHO

N

NN

N

O

O

O

HN O

Acetanilide Caffeine

Acetylsalicylic acid

2-Acetamidophenol


1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

1

2

3

4

5V0 = 1.37

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 100 mM HCOONH4, pH 3.75Flow Rate: 1.4 mL/minGradient: Time Profile

(min) %A %B %C0.0 65 25 105.0 40 50 1010.0 40 50 10


Compounds: USP Tailing1. Procaine 1.072. Lidocaine 1.043. Butacaine 1.044. Benzocaine 0.985. Tetracaine 1.20

N

O

O

H2N

NH2

O O

NH

O

N

Benzocaine Butacaine

Lidocaine

O

O

N

H2N

Procaine

Tetracaine

HN

O

O

N

Anesthetics


Arbutin

V0 = 0.98

USP Tailing = 0.96

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Flow Rate: 2.0 mL/minIsocratic Mobile Phase Composition: 10 mM HCOONH4, pH 3.75Injection Volume: 5.0 µLTemperature: 30°CDetection: UV @ 280 nmInstrument: Alliance® 2695, 2996 PDA

Arbutin


1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

1

2

3

4

5

6

V0 = 0.98

Conditions


(min) %A %B %C0.0 90 0 1010.0 65 25 10


Compounds: USP Tailing1. Gallic acid 0.872. Protocatechuic acid 1.033. Gentisic acid 1.024. 4-Hydroxybenzoic acid 1.105. Vanillic acid 1.086. Syringic acid 1.09

Aromatic Acids

O H

O

OH

4-Hydrobenzoic Acid Gallic Acid

Vanillic Acid

OH

O

O H

O H

O H

O H

O

O H

OHOH

O

O H

O H

O

OH

O

O

OH

O

O HO

O H

Syringic acid

Gentisic acid Protocatechuic acid


Vo= 1.18

1

2

Aromatic Amino Acids

V0 = 1.18

3

1

2

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Conditions


(min) %A %B %C0.0 90 0 1010.0 10 80 10


Compounds: USP Tailing1. Tyrosine 1.182. Phenylalanine 1.133. Tryptophan 1.11

NH2

OH

O

Phenylalanine Tyrosine

NH2

HO

OH

O

O

OH

NH2HN

Tyrptophan



Atlantis™ 100% Aqueous QC Batch Test

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

V0 = 1.20

1

2

34

5

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Flow Rate: 1.2 mL/minIsocratic Mobile Phase Composition: 10 mM HCOONH4, pH 3.0Injection Volume: 7.0 µLTemperature: ambientDetection: UV @ 254 nmInstrument: Alliance® 2690, 2996 PDA

Compounds: USP Tailing1. Thiourea 1.112. 5-Fluorocytosine 1.093. Adenine 1.134. Guanosine-5’-monophosphate 1.115. Thymine 1.03

5-Fluorocytosine Adenine

Guanosine-5-monophosphateThiourea

Thymine

β-Blockers

V0 = 0.98

31

2

4

56

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Compounds: USP Tailing1. Atenolol 1.022. Pindolol 1.063. Metoprolol 1.134. Oxprenolol 1.325. Labetolol 1.316. Propranolol 1.41

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4 , pH 3.0Flow Rate: 2.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 85 5 103.0 65 25 1010.0 65 25 10


O

OH

NH

NH2

O

Atenolol

HN

HO

OH

NH2

O

O

O

OH

NH

NH

OH

O

O

OH

ONH NH

O

HO

NH

Labetolol

Metoprolol

PindololPropranolol

Oxprenolol



Catechins

V0 = 1.83

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Minutes

1 3

4

2

56

7

8

9

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 1% HCOOH, pH 2.3Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 70 20 1010.0 60 30 1020.0 30 60 10


Compounds: USP Tailing1. Gallic Acid 1.052. Epigallocatechin 0.943. Catechin 1.004. Caffeine 0.975. Epigallocatechin gallate 1.086. Epicatechin 0.987. Gallocatechin gallate 0.988. Epicatechin gallate 0.989. Catechin gallate 0.99

N

NN

N

O

O

Caffeine

OH

O

O H

O HGallic acid

Epigallocatechingallate

Epicatchin

Epigallocatechin

Epicatechingallate

Catechingallate

Catechin

Gallocatechin gallate

Catecholamines - Isocratic

V0 = 1.83

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Minutes

3

1 2

4

56

7

Compounds: USP Tailing1. Norepinephrine (NE) 1.212. Epinephrine (E) 1.203. Dopamine (DA) 1.214. 3,4-Dihydroxyphenylacetic acid (DOPAC) 1.005. Serotonin (5-HT) 1.106. 5-Hydroxy-3-indole acetic acid (5-HIAA) 0.977. 4-Hydroxy-3-methoxyphenylacetic acid (HVA) 0.97

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Composition: 88% A; 2% B; 10% CInjection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 280 nmInstrument Alliance® 2695, 2996 PDA

DOPAC

O

OH

HO

HO

Epinephrine

HO

HO

OH

HN

HVAO

HO

O

OH

Serotonin

HN

NH2

HO

VMA

OH

O

OHO

HO

5-HIAA

NH

O

OH

HO

O

OH

NH2

HO

HO

DOPA



Catecholamines - Gradient

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Minutes

V0 = 1.83

1

9

2

34 5

6

7 810

Conditions


(min) %A %B %C0.0 90 0 105.0 90 0 1015.0 65 25 1020.0 65 25 10


Compounds: USP Tailing1. Norepinephrine (NE) 1.162. Epinephrine (E) 1.173. 3,4-Dihydroxyphenylalanine (DOPA) 1.154. Dopamine (DA) 1.045. 3-Methoxy-4-hydroxyphenylglycol (MHPG) 1.166. 4-Hydroxy-3-methoxymandelic acid (VMA) 1.177. Serotonin (5-HT) 1.108. 3,4-Dihydroxyphenylacetic acid (DOPAC) 1.099. 5-Hydroxy-3-indole acetic acid (5-HIAA) 1.1110.4-Hydroxy-3-methoxyphenylacetic acid (HVA) 1.12

5-HIAA

NH

O

OH

HO

DOPAC

O

OH

NH2

HO

HO

O

OH

HO

HO

DOPA

NH2HO

HO

Dopamine

Epinephrine

HO

HO

OH

HN

HVAO

HO

O

OH

MHPG

O

HO

OH

OH

Norepinephrine

HO

HO

OH

NH2

Serotonin

HN

NH2

HO

VMA

OH

O

OHO

HO

Cold Medicine Products

V0 = 1.83

1

2

3

45 6

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Compounds: USP Tailing1. Acetaminophen 1.042. Pseudoephedrine 1.113. Chlorpheniramine 1.044. Guaiacol glyceryl ether 1.015. Dextromethorphan 1.006. Diphenhydramine 1.08

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: 0.1% TFAMobile Phase B: ACNFlow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.0 77 231.0 77 233.0 52 4810.0 52 48


HN

O

OH

Acetaminophen

N

N

Cl

Chlorpheniramine

O

H

N

Dextromethorphan

O

N

Diphenhydramine

O

O

OH

OH

Guaiacol glyceryl ether

NH

OH

Pseudophendrine



Cytochrome C Tryptic Digest

Scan ESI+: TIC1.00e9

%

5.00 10.00 15.00 20.00 25.00 30.00Minutes

0 Time

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µm Part Number: 186001329Mobile Phase A: 0.1 % HCOOHMobile Phase B: 0.065 % HCOOH in ACNFlow Rate: 0.75 mL/min, 0.2 mL/min Split to MSGradient: Time Profile

(min) %A %B0.0 100 045.0 60 40

Injection Volume: 20.0 µLInjection Mass: 20.0 µgTemperature: ambientInstrument: Alliance® HT 2795

Waters ZQ Settings:

Source: ESI (+)Capillary (kV): 3.5Cone (V): 30Temperature

Source: 150°C Desolvation: 350°C

Gas FlowCone: 50L/HrDesolvation: 500L/Hr

m/z: 300-1500

Diuretics

V0 = 1.37

3

1

2

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Compounds: USP Tailing1. Hydrochlorothiazide 0.942. Chlorthalidone 0.993. Furosemide 0.99

Conditions


(min) %A %B %C0.0 67 23 1010.0 35 55 10


Chlorthalidone

Furosemide

Hydrochlorothiazide

©2002 Waters Corporation. Waters, Atlantis,and Alliance are trademarks of Waters Corporation 21

22 ©2002 Waters Corporation. Waters, Atlantis,and Alliance are trademarks of Waters Corporation

Drugs for Rhinitis

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

V0 = 1.37

1 2

3

4

5

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 1.0% HCOOH, pH 2.3Flow Rate: 1.4 mL/minGradient: Time Profile

(min) %A %B %C0.0 90 0 10

10.0 50 40 10Injection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing1. Phenylephrine 1.102. Maleic acid 1.173. Phenylpropanolamine 1.264. Caffeine 1.055. Chlorpheniramine 1.26

N

NN

N

O

O

Caffeine

N

N

Cl

Chlorpheniramine

Maleic acid

Phenylephrine

NH2

OH

Phenylpropanolamine

Ginsenosides

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

V0 = 1.83

1

2

34

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNFlow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.0 75 254.0 60 4010.0 60 40


Compounds: USP Tailing1. Ginsenoside Rg1 1.022. Ginsenoside Rc 1.013. Ginsenoside Rb2 1.134. Ginsenoside Rd 0.99



Lipid Soluble Antioxidants

V0 = 0.98

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

1 2

3

4

5

6

7

8

Conditions


(min) %A %B %C %D0.0 55 17.5 10 17.54.0 35 27.5 10 27.56.0 0 45 10 4515.0 0 45 10 45


Compounds: USP Tailing1. Propyl Gallate (PG) 1.082. 2,4,5-Trihydroxybutyrophenone (THBP) 1.093. Tert-butylhydroquinone (TBHQ) 1.014. Butylated hydroxyanisole (BHA) 1.025. 2,6-Di-tert-butyl-4-hydroxymethylphenol 1.066. Octyl Gallate (OG) 1.117. Butylated hydroxytoluene (BHT) 0.958. Lauryl Gallate (LG) 1.16

PG

THBP

TBHQ BHA

2,6-Di-ter-butyl-4-hydroxymethylhphenol

OG

LG

BHT

Nalidixic Acid Antibiotics - Isocratic

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

V0 = 0.50

1

23

Compounds: USP Tailing1. Cinoxacin 1.252. Oxolinic acid 1.153. Nalidixic acid 1.03

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µmPart Number: 186001329Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: ACNMobile Phase D: 1% HCOOH, pH 2.23Flow rate: 2.0 mL/minIsocratic Mobile Phase Composition: 55% A;30% B;5% C; 10% DInjection Volume: 5µLSample concentration: 10 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA

Cinocaxin

Nalidixic acid

Oxolinic acid



Nalidixic Acid Antibiotics - Gradient

V0 = 0.50

1

2

3

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

Compounds: USP Tailing1. Cinoxacin 1.102. Oxolinic acid 1.083. Nalidixic acid 1.08

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µmPart Number: 186001329Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 1% HCOOH, pH 2.23Flow rate: 2.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 70 20 1010.0 10 80 10

Injection Volume: 5 µLSample concentration: 10 µg/mLTemperature: 30°CDetection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA

Cinocaxin

Nalidixic acid

Oxolinic acid

Nalidixic Acid Antibiotics – LC/MS

21

3

%

100

%

0100

-16

Scan ES+ TIC5.35e9

Diode Array254nm5.22e4

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

Conditions

Column: Atlantis™ 2.1 x 30 mm, 3 µmPart Number: 186001287Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 1% HCOOH in H2OFlow rate: 0.8 mL/min with 0.2 mL/min to MSGradient: Time Profile

(min) %A %B %C0.0 75 15 105.0 40 50 10

Injection Volume: 10 µLTemperature: 30°CDetection: UV @ 254 nm and MS ESI+Instrument: Alliance® HT 2795, 2996 PDA

Waters ZQ Settings:Source: ESI+Capillary (kV) 3.5Cone (V) 50Extractor 3.0RF Lens 0.1Source Temp 150Desolvation Temp 400Cone gas Flow (L/Hr) 50Desolvation Gas Flow (L/Hr) 500LM Resolution 15HM Resolution 15Ion Energy 0.5Multiplier (V) 650

Compounds: MW1. Cinoxacin 262.222. Oxolinic acid 261.233. Nalidixic acid 232.24

Cinocaxin

Nalidixic acid

Oxolinic acid



Nalidixic Acid Antibiotics – LC/MS

%

100 Scan ES+ 262

8.66e8

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

0

%

100

0

%

100

0

%

100

0

Oxolinic acid

Nalidixic acid

Cinoxacin

Scan ES+ 233

9.55e8

Scan ES+ 263

3.33e8

Scan ES+ TIC

5.35e9

Compounds: MW1. Cinoxacin 262.222. Oxolinic acid 261.233. Nalidixic acid 232.24

CinocaxinNalidixic acidOxolinic acid

Nucleic Acids

V0 = 1.83

1

2 3

4

5 6

7

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Compounds: USP Tailing1. Cytosine 1.112. 5-Fluorocytosine 1.083. Uracil 1.054. 5-Fluorouracil 1.185. Guanine 1.166. Thymine 1.187. Adenine 1.25

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 90 0 1010.0 84 6 10


Cytosine5-Fluorocytosine

Uracil 5-Fluorouracil

Guanine Thymine

Adenine



Nucleosides

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

V0 = 1.37

1

2

3

4

67

8

Conditions


(min) %A %B %C0.0 90 0 1010.0 82 8 10


Compounds: USP Tailing1. Cytidine 1.052. 2’-Deoxycytidine 1.023. Uridine 1.084. Adenosine 1.075. 2’-Deoxyadenosine 1.086. Guanosine 1.097. 2’-Deoxyguanosine 1.108. Thymidine 1.10

Cytidine 2-Deoxycytidine

Urdine

2-Deoxyadenosine

2-Deoxyguanosine

Guanosine

Thymidine Adenosine

5

Organic Acids

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Minutes

1

3

24

5

6

78

9

V0 = 3.51

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Flow Rate: 0.5 mL/minIsocratic Mobile Phase Composition: 20 mM NaH2PO4, pH 2.7Injection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 210 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing1. Oxalic acid 1.352. Pyruvic acid 1.123. Tartaric acid 1.134. Malic acid 1.195. Lactic acid 1.246. Acetic acid 1.307. Maleic acid 1.248. Fumaric acid 1.239. Succinic acid 1.26

Oxalic acid

Pyruvic acid

Tartaric acid

Maleic acid

Lactic acid

OH

O

Acetic acid

Malic acid

Fumaric acid

Succinic acid



Oxidative Stress Markers – 5% ACN, pH 3.0

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

1

V0 = 1.37

2

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0Flow Rate: 1.4 mL/minIsocratic MobilePhase Composition: 85% A; 5% B; 10% CInjection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 280 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing1. 2’-Deoxyguanosine 1.042. 8-Hydroxy-2’-deoxyguanosine 0.99

2- Deoxyguanosine

8- Hydroxy-2-Deoxyguanosine

Oxidative Stress Markers - 5% MeOH, pH 5.0

1

2

V0 = 1.83

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

Minutes

Conditions

Column: Atlantis™ 4.6 x 100 mm, 3 µm Part Number: 186001337Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Conditions: 85% A; 5% B; 10% CInjection Volume: 10.0 µLTemperature: 30°CDetection: UV @ 285 nmInstrument: Alliance® 2695, 2996 PDA


2- Deoxyguanosine




Oxidative Stress Markers - 10% MeOH, pH 5.0

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

1

2

V0 = 1.83

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 100 mM CH3COONH4, pH 5.0Flow Rate: 1.0 mL/minIsocratic Mobile Phase Conditions: 80% A; 10% B; 10% CInjection Volume: 20.0 µLTemperature: 30°CDetection: UV @ 285 nmInstrument: Alliance® 2695, 2996 PDA


2- Deoxyguanosine


Paclitaxel

USP Tailing = 1.01

V0 = 1.38

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0 Flow Rate: 1.4 mL/minIsocratic Mobile Phase Conditions: 30% A; 60% B; 10% CInjection Volume: 2.0 µLTemperature: 30°CDetection: UV @ 238 nmInstrument: Alliance® 2695, 2996 PDA

Paclitaxel



Peptides

0 4 8 12 16 20Minutes

V0 = 0.79 12

3

4 56

7

8

9

Compounds:1. L22752. A66773. Bradykinin4. Angiotensin II5. Angiotensin I6. Substance P7. Renin Substrate8. Insulin B Chain9. Melittin

Conditions

Column: Atlantis™ 4.6 x 50 mm, 5 µm Part Number: 186001331Mobile Phase A: 0.02% TFA in H2OMobile Phase B: 0.016% TFA in ACNFlow Rate: 0.75 mL/minGradient: Time Profile

(min) %A %B0.0 100 025.0 50 50

Injection Volume: 20 µLTemperature: 40°CTotal Mass Load: 18 µgDetection: UV @ 220 nmInstrument: Alliance® 2690, 2996 PDA

Phthalic Acids

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Minutes

1

2

3

V0 = 1.37

Conditions


(min) %A %B %C0.0 55 35 105.0 30 60 10


Compounds: USP Tailing1. Phthalic acid 1.102. Terephthalic acid 1.033. Isophthalic acid 1.00

Phthalic acid

Terphthalic acid

Isophthalic acid



Phthalic Esters

1

2

3

V0 = 1.83

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

Compounds: USP Tailing1. Dimethyl phthalate 1.062. Dibutyl phthalate 1.043. Dioctyl phthalate 0.99

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: ACNFlow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.0 10 904.0 0 10010.0 0 100


Dibutyl phthalate

Dimethyl phthalate

Dioctyl phthalate

Polar and Non-Polar Compounds Test Mix: 0-100% ACN, 30 mm column

V0 = 0.20

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00

Minutes

1

2

3 4

5

6

7

89

10

Conditions

Column: Atlantis™ 2.1 x 30 mm, 3 µm Part Number: 186001287Mobile Phase A: 0.1% HCOOH in H2OMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 0.8 mL/minGradient: Time Profile

(min) %A %B0.0 100 00.2 100 03.2 0 1004.0 0 100

Injection Volume: 2.0 µLTemperature: ambientDetection: UV @ 254 nmSampling Rate: 10 pts/secInstrument: Alliance® HT 2795, 2996 PDA

Compounds: USP Tailing1. 8-bromoguanosine 1.152. Acetanilide 1.013. Triamcinolone 0.994. Hydrocortisone 1.015. 2-Amino-7-chloro-5-oxo-5H-

[1]benzopyrano[2,3-b]pyridine-3-carbonitrile 1.02

6. 6α-Methyl-17α-hydroxyprogesterone 0.967. 3-Aminofluoranthene 1.018. 2-Bromofluorene 1.039. Perylene 1.0110. Naphtho(2,3-a)pyrene 1.03

Triamcinolone

3-Aminofluoranthene

Acetanilide

2-Bromofluorene

Perylene

Naphtho(2,3-a)pyrene

2-Amino-7chloro-5-oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carbonitrile

Hydrocortisone

6α-Methyl-17α-hydroxyprogesterone

8-Bromoguanosine



Polar and Non-Polar Compounds Test Mix: 0-100% ACN, 150 mm column

1

2

3 45

6

7

8

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

V0 = 0.98

9

10

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: 0.1% HCOOH in H2OMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 2.0 mL/min Gradient: Time Profile

(min) %A %B0.0 100 00.5 100 010 0 10020 0 100

Injection Volume: 10.0 µLTemperature: ambientDetection: UV @ 254 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing1. 8-bromoguanosine 1.102. Acetanilide 1.023. Triamcinolone 1.084. Hydrocortisone 0.955. 2-Amino-7-chloro-5-oxo-5H-



Triamcinolone

3-Aminofluoranthene

Acetanilide

2-Bromofluorene

Perylene



Hydrocortisone


8-Bromoguanosine

Polar and Non-Polar CompoundsTest Mix: (with Uracil), 0-100% ACN, 150 mm column

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

V0 = 0.98

1 2 3

4 56

7

8 9

10

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: 0.1% HCOOH in H2OMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 2.0 mL/minGradient: Time Profile

(min) %A %B0.0 100 00.5 100 010 0 10020 0 100

Injection Volume: 10.0 µLTemperature: ambientDetection: UV @ 254 nmInstrument: Alliance® 2695, 2996 PDA

Compounds: USP Tailing

1. Uracil 1.102. Acetanilide 1.023. Triamcinolone 1.074. Hydrocortisone 0.945. 2-Amino-7-chloro-5-oxo-5H-


6. 6α−Methyl-17α-hydroxyprogesterone 0.987. 3-Aminofluoranthene 1.008. 2-Bromofluorene 0.989. Perylene 0.9810. Naphtho(2,3-a)pyrene 1.00

Uracil

Triamcinolone

3-Aminofluoranthene

Acetanilide

2-Bromofluorene

Perylene



Hydrocortisone


8-Bromoguanosine



Polar and Non-Polar Compounds Test Mix: (with Uracil) 10-95% ACN, 150 mm column

V0 = 0.98

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

1

2 3

4 56

7

8 9

10

Conditions


(min) %A %B %C0.0 80 10 1010.0 0 95 515.0 0 95 5


Compounds: USP Tailing1. Uracil 1.042. Acetanilide 0.953. Triamcinolone 1.024. Hydrocortisone 1.035. 2-Amino-7-chloro-5-oxo-5H-



Uracil

Triamcinolone

3-Aminofluoranthene

Acetanilide

2-Bromofluorene

Perylene



Hydrocortisone


8-Bromoguanosine


Polar Pesticides

AcephateMethamidophos

2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time0

100

%

Conditions

Column: Atlantis™ 2.1 x 100 mm, 3 µm Part Number: 186001295Mobile Phase A: H2OMobile Phase B: ACNFlow Rate: 0.17 mL/minIsocratic MobilePhase Composition: 95% A; 5% BInjection Volume: 20.0 µLTemperature: 25˚CDetection: MS(ESI+)Instrument: Alliance® 2795

Waters ZMDESI+Capillary (kV) 3.1Extractor 3.0RF Lens 0.1Source Temp 150Desolvation Temp 350Desolvation Gas Flow (L/Hr) 500LM Resolution 12.5HM Resolution 12.5Ion Energy 1.0Multipler (V) 650

SIR of 5 Massesmass (m/z) cone (V)112a 40113b 40142a 20143b 25184b 15

aion for methamidophosbion for acephate

2

1

Compounds:1.Methamidophos2.Acephate

Minutes

Purine Alkaloids

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

V0 = 0.98

1

2

3

4

Compounds: USP Tailing1. Theobromine 1.002. Theophylline 1.003. Caffeine 0.984. Phenol 0.97

Conditions


(min) %A %B %C0.0 85 5 105.0 50 40 10


TheobromineTheophylline

Caffeine

N

NN

N

O

O

Phenol


Sulfonamides

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Minutes

V0 = 0.98

1 2

3

4

5

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: H2OMobile Phase B: MeOHMobile Phase C: 1% HCOOH, pH 2.4Flow Rate: 2.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 75 15 1010.0 50 40 10


Compounds: USP Tailing1. Sulfadiazine 1.012. Sulfathiazole 1.023. Sulfamerazine 1.004. Sulfamethazine 0.995. Sulfamethoxazole 0.99

Sulfadiazine

Sulfamerazine

Sulfadmethazine Sulfamethoxazole

Sulfathiazole


Vancomycin

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

Minutes

V0 = 0.51USP Tailing = 1.03

Conditions

Column: Atlantis™ 4.6 x 50 mm, 3 µmPart Number: 186001329Mobile Phase A: H2OMobile Phase B: ACNMobile Phase C: 100 mM HCOONH4, pH 3.0Flow rate: 2.0 mL/minGradient: Time Profile

(min) %A %B %C0.0 90 0 105.0 78 12 10

Injection Volume: 10 µLSample Concentration: 40 µg/mLTemperature: 30°C Detection: UV @ 254 nmInstrument: Alliance® HT 2795, 2996 PDA

Vancomycin


Water Soluble Vitamins

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Minutes

12

3

4

5 6

78

V0 = 1.37

Compounds: USP Tailing1. L-ascorbic acid 1.122. Nicotinic acid 1.273. Thiamine 1.204. Pyridoxal 1.135. Pyridoxine 1.046. Folic acid 1.107. Caffeine 1.108. Riboflavin 1.14

Conditions

Column: Atlantis™ 4.6 x 150 mm, 5 µm Part Number: 186001344Mobile Phase A: 0.1% TFAMobile Phase B: ACNFlow Rate: 1.4 mL/minGradient: Time Profile

(min) %A %B0.0 100 04.0 97 36.0 85 1515.0 80 20


L-Ascorbic acid Nicotinic acid Pyridoxine

Caffeine

N

NN

N

O

O

Pyridoxal

Thiamine

Thiamine

Riboflavin


Column Care and Use Instructions

PLEASE READ CAREFULLY BEFORE USING ATLANTIS™ COLUMNS.

The bonding processes for Atlantis™ packing materials result in HPLC columns with minimal column bleed and maximum hydrolyticstability at low pH. Atlantis™ columns are designed to operate in 100% aqueous mobile phases without dewetting. In fact, each batch ofAtlantis™ material is tested chromatographically using a 100% aqueous mobile phase. The manufacture of Atlantis™ columns begins withultrapure reagents to control the chemical composition and purity of the final product. Atlantis™ columns are manufactured in a cGMP,ISO 9002 certified plant with each step being conducted within narrow tolerances. Every column is individually tested and Certificatesof Batch Analysis and a Performance Chromatogram are provided with each column.

Waters recommends the use of Waters Sentry™ guard columns to maximize column lifetime and protect the column from contaminants.

Connecting the Column or Cartridge to the HPLC

Column Connection

Handle the column with care. Do not drop or hit the column on a hard surface as it may disturb the bed and affect its performance.

1. Correct connection of 1/16 inch outer diameter stainless steel tubing leading to and from the column is essential for high-quality chromatographic results.

2. When using standard stainless steel compression screw fittings, it is important to ensure proper fit of the 1/16 inch outer diameter stainless steel tubing. When tightening or loosening the compression screw, place a 5/16 inch wrench on the compression screw and a 3/8 inch wrench on the hex head of the column endfitting.

Note: If one of the wrenches is placed on the column flat during this process, the endfitting will be loosened and leak.

3. If a leak occurs between the stainless steel compression screw fitting and the column endfitting, a new compression screw fitting, tubing and ferrule must be assembled.

4. An arrow on the column identification label indicates correct direction of solvent flow.

Figure 1: Installation of Atlantis™ Cartridge Column with Sentry™ Guard Column

0.25 mm (0.009 inch) Tubing Atlantis™ Cartridge Column End Connector

Sentry™ GuardColumn

Spacer C-Clip

Figure 2: Waters and Parker Ferrule Types

Waters Ferrule Setting Parker Ferrule Setting

Figure 3: Proper Tubing/Column ConnectionTubing touches the bottom of the column endfitting, with no void between them.


Cartridge Connection

Handle the cartridge with care. Do not drop or hit the cartridge on a hard surface as it may disturb the bed and affect itsperformance. Refer to Figure 1 for an exploded view of an Atlantis™ cartridge column with a Sentry™ guard column.

1. Unscrew end connectors from the old cartridge. Leave them connected to the inlet and outlet lines of the instrument.

2. Attach new cartridge column between connectors so that the direction of the flow arrow on the label is pointing in the direction of mobile phase flow (toward detector).

3. Finger tighten all fittings.

It is important to realize that extra column peak broadening can destroy a successful separation. The choice of appropriate columnconnectors and system tubing is discussed in detail below.

Column Connectors and System Tubing Considerations

Due to the absence of an industry standard, various column manufacturers have employed different types of chromatographic columnconnectors. The chromatographic performance of the separation can be negatively affected if the style of the column endfittings does notmatch the existing tubing ferrule setting. This section explains the differences between Waters style and Parker style ferrules and endfittings(Figure 2). Each endfitting style varies in the required length of the tubing protruding from the ferrule. The Atlantis™ column is equipped withWaters style endfittings which require a 0.130 inch ferrule. If a non-Waters style column is presently being used, it is critical that ferruledepth be reset for optimal performance prior to installing an Atlantis™ column.

In a proper tubing/column connection (Figure 3), the tubing touches the bottom of the column endfitting, with no void between them.

The presence of a void in the flow stream reduces column performance. This can occur if a Parker ferrule is connected to a Waters styleendfitting (Figure 4).

Note: A void appears if tubing with a Parker ferrule is connected to a Waters style column.

Figure 4: Parker Ferrule in a Waters Style Endfitting

There is only one way to fix this problem: Cut the end of the tubing with the ferrule, place a new ferrule on the tubing and make a newconnection. Before tightening the screw, make sure that the tubing bottoms out in the endfitting of the column.

Conversely, if tubing with a Waters ferrule is connected to a column with Parker style endfitting, the end of the tubing will bottom out beforethe ferrule reaches its proper sealing position. This will leave a gap and create a leak (Figure 5).

Note: The connection leaks if a Waters ferrule is connected to a column with a Parker style endfitting.

Figure 5: Waters Ferrule in a Parker Style Endfitting

There are two ways to fix the problem:

1. Tighten the screw a bit more. The ferrule moves forward, and reaches the sealing surface. Do not over tighten since this may end in breaking the screw.

2. Cut the tubing, replace the ferrule and make a new connection.


Alternatively, replace the conventional compression screw fitting with an all-in-one PEEK fitting (Waters part number PSL613315) that allowsresetting of the ferrule depth. Another approach is to use a Keystone, Inc. SLIPFREE® connector to always ensure the correct fit. The finger-tight SLIPFREE® connectors automatically adjust to fit all compression screw type fittings without the use of tools (Figure 6).

Figure 6: Single and Double SLIPFREE® Connectors

SLIPFREE® Connectors Features

• Tubing pushed into endfitting, thereby guaranteeing a void-free connection

• Connector(s) come(s) installed on tubing

• Various tubing IDs and lengths available

• Fingertight to 10,000 psi – Never needs wrenches

• Readjusts to all column endfittings

• Compatible with all commercially available endfittings

• Unique design separates tube-holding function from sealing function

Table 1. Waters Part Numbers for SLIPFREE® Connectors

Band Spreading Minimization

Figure 7 shows the influence of tubing internal diameter on system band spreading and peak shape. As can be seen, the larger tubingdiameter causes excessive peak broadening and lower sensitivity.

Figure 7: Effect of Connecting Tubing on System

SLIPFREE® Type and Tubing Length Tubing Internal Diameter

0.005” 0.010” 0.020”Single 6 cm PSL 618000 PSL 618006 PSL 618012Single 10 cm PSL 618002 PSL 618008 PSL 618014Single 20 cm PSL 618004 PSL 618010 PSL 618016Double 6 cm PSL 618001 PSL 618007 PSL 618013Double 10 cm PSL 618003 PSL 618009 PSL 618015Double 20 cm PSL 618005 PSL 618001 PSL 618017

Diluted/Distorted Sample Band

0.005 inches

0.020 inches

0.040 inches

0.005 inches

0.020 inches

0.040 inches

Diluted/Distorted Sample Band

50 ©2002 Waters. Corporation Waters and Atlantis are trademarks of Waters Corporation

Measuring System Bandspread Volume

This test should be performed on an HPLC system with a single wavelength UV detector (not a Photodiode Array (PDA)).

1. Disconnect column from system and replace with a zero dead volume union.

2. Set flow rate to 1 mL/min.

3. Dilute a test mix in mobile phase to give a detector sensitivity 0.5 - 1.0 AUFS (system start up test mix can be used which contains uracil, ethyl and propyl parabens; Waters part number WAT034544).

4. Inject 2 to 5 µL of this solution.

5. Using 5 sigma method measure the peak width at 4.4% of peak height:

Band Spread (µL) = Peak Width (min) x Flow Rate (µL/min)

= 0.1 min x 1000 µL/min

= 100 µL

Figure 8: Determination of System Bandspread Volume Using 5-Sigma Method

In a typical HPLC system, the Bandspread Volume should be 100 µL ± 30 µL.

In a microbore (≤2.1mm i.d.) system, the Bandspread Volume should be no greater than 20 to 40 µL.

Measuring Gradient Delay Volume

1. Replace the column with a zero dead volume union.

2. Prepare eluent A (pure solvent, such as methanol) and eluent B (solvent plus sample, such as 5.6 mg/mL propylparaben in methanol).

3. Equilibrate the system with eluent A until a stable baseline is achieved.

4. Switch to 100% eluent B.

5. Record the half height of the step and determine dwell volume (Figure 9).

Figure 9: Determination of Dwell Volume

The dwell volume should be less than 1 mL. If the dwell volume is greater than 1 mL, see System Modification Recommendationssection on how to reduce system volume.

Volume (µL)

Dwell�Volume

1.0

0.8

0.6

0.4

Height

0.2

0.0


Use of Narrow-Bore (≤3.0 mm i.d.) Columns

This section describes how to minimize extra column effects and provides guidelines on maximizing the performance of a narrow-bore column in an HPLC system. A 3.0 mm i.d. narrow-bore column usually requires no system modifications. A 2.1 mm i.d.column, however, requires modifications to the HPLC system in order to eliminate excessive system bandspread volume. Withoutproper system modifications, excessive system bandspread volume causes peak broadening and has a large impact on peak widthas peak volume decreases.

Impact of Bandspread Volume on 2.1 mm i.d. Column Performance

System with 70 µL bandspread: 10,000 plates

System with 130 µL bandspread: 8,000 plates (same column)

Note: Flow splitters after the column will introduce additional bandspreading.

System optimization, especially in a system that contains a flow splitter, can have dramatic effects on sensitivity and resolution. Optimizationincludes using correct-depth ferrules and minimizing tubing diameter and lengths. An example is given in Figure 10 where systemoptimization resulted in a doubling of sensitivity and resolution of the metabolite in an LC/MS/MS system.

Figure 10: Non-Optimized vs. Optimized LC/MS/MS System

System Modification Recommendations

1. Use a microbore detector flowcell with ≤2.1 mm i.d. columns.

Note: Detector sensitivity is reduced with the shorter flowcell pathlength in order to achieve lower bandspread volume.

2. Minimize injector sample loop volume.

3. Use 0.009 inch (0.25 mm) tubing between pump and injector.

4. Use 0.009 inch (0.25 mm) tubing for rest of connections in standard systems and 0.005 inch (0.12 mm) tubing for narrowbore (≤2.1 mm i.d.) systems.

5. Use perfect (pre-cut) connections (with a variable depth inlet if using columns from different suppliers).

6. Detector time constants should be shortened to less than 0.2 seconds.

Waters Small Particle Size (3 µm) Columns – Fast Chromatography

Waters columns that contain 3 µm particles provide faster and more efficient separations without sacrificing column lifetime. This sectiondescribes five parameters to consider when performing separations with columns containing 3 µm particles.

Note: Columns that contain 3 µm particles have smaller outlet frits to retain packing material. These columns should not be backflushed.

1. Flow Rate — Compared with the 5 µm columns, columns with 3 µm particles have higher optimum flow rates. These columns are used when high efficiency and short analysis times are required. These higher flow rates, however, lead to increased backpressure.

Note: Use a flow rate that is practical for your system.

2. Backpressure — Backpressures for columns with 3 µm particles are higher than for 5 µm columns with the same dimensions. Waters suggests using a shorter column to compensate for increased backpressure and to obtain a shorter analysis time.

Non-optimized LC/MS/MS System Optimized System

7.00 7.50 8.00 7.00 7.50 8.00


3. Temperature — Use a higher temperature to reduce backpressure caused by smaller particle sizes. The recommended temperature range for Atlantis™ columns is 20°C to 45°C. See Column Usage section for a discussion of elevated temperature use with Atlantis™ columns.

4. Sampling Rate — Use a sampling rate of about 10 points per second.

5. Detector Time Constant — Use a time constant of 0.1 seconds for fast analysis.

Column Performance Validation

Each Atlantis™ column comes with Certificates of Batch Analysis and a Performance Test Chromatogram. The Certificates of Analysis arespecific to each batch of packing material contained in the Atlantis™ column and includes the gel lot batch number, analysis of unbondedparticles, analysis of bonded particles, and chromatographic results and conditions. The Performance Test Chromatogram is specific toeach individual column and contains information such as gel lot batch number, column serial number, USP plate count, USP tailing factor,capacity factor and chromatographic results and conditions. These data should be stored for future reference.

Initial Column Efficiency Determination

1. Perform an efficiency test on the column before using it. Waters recommends using a suitable solute mixture, as found in the “Performance Test Chromatogram”, to analyze the column upon receipt.

2. Determine the number of theoretical plates (N) and use for periodic comparison.

3. Repeat the test periodically to track column performance over time. Slight variations may be obtained on two different HPLC systems due to the quality of the connections, operating environment, system electronics, reagent quality, column condition and operator technique.

Column Equilibration

Atlantis™ columns are packed and shipped in 100% acetonitrile. It is important to ensure solvent compatibility before changing to a newsolvent. Equilibrate the column with a minimum of 10 column volumes of the mobile phase to be used (refer to Table 2 for a listing ofstandard column volumes).

Table 2. Standard column volumes in mL (multiply by 10 for flush solvent volumes)

Column Installation Procedure

Note: The flow rates given in the procedure below are for a typical 4.6 mm i.d. column. Scale the flow rate up or down accordinglybased upon the column i.d., length, particle size and backpressure of the Atlantis™ column being installed. See Scaling Up/Downsection for calculating flow rates when changing column i.d and/or length.

1. Purge the pumping system and connect the inlet end of the column to the injector outlet.

2. Set the pump flow to 0.1 mL/min. and increase to 1 mL/min over 5 minutes.

Column Volume (mL)

Column Column internal diameter (mm)Length 1.0 2.1 3.0 3.9 4.6 7.8 10 19 30 50

30 mm – 0.1 0.2 – 0.5 – 2.4 8 – –

50 mm 0.1 0.2 0.3 – 0.8 2.4 4 14 35 98

100 mm 0.1 0.4 0.7 1.2 1.7 5 8 28 70 –

150 mm 0.1 0.5 1.0 1.8 2.5 7 12 42 106 294

250 mm – 0.9 1.8 – 4 – 20 70 176 490

300 mm – – – – – 14 24 85 212 589


3. When the mobile phase is flowing freely from the column outlet, attach the column to the detector. This prevents entry of air intothe detection system and gives more rapid baseline equilibration.

4. When the mobile phase is changed, gradually increase the flow rate of the new mobile phase from 0.0 mL/min to 1.0 mL/min in 0.1 mL/min increments.

5. Once a steady backpressure and baseline have been achieved, the column is ready to be used.

Note: If mobile phase additives are present in low concentrations (e.g., ion-pairing reagents), 100 to 200 column volumes may berequired for complete equilibration. In addition, mobile phases that contain formate (e.g., ammonium formate, formic acid, etc.) mayalso require slightly longer initial column equilibration times.

Column Usage

To ensure the continued high performance of Atlantis™ columns and cartridges, follow these guidelines:

Guard columns

Use a Waters Sentry™ guard cartridge of matching chemistry and particle size between the injector and main column. It is important to usea high-performance matching guard column to protect the main column while not compromising or changing analytical resolution.

Sample Preparation

1. Sample impurities often contribute to column contamination. One way to avoid this is to use Waters Oasis® solid-phase extraction cartridges/columns or Sep-Pak® cartridges of the appropriate chemistry to clean up the sample before analysis.

2. It is preferable to prepare the sample in the mobile phase or a solvent that is weaker (less organic modifier) than the mobile phase.

3. If the sample is not dissolved in the mobile phase, ensure that the sample, solvent and mobile phases are miscible in order to avoid sample and/or buffer precipitation.

4. Filter sample with 0.2 µm membrane to remove particulates. If the sample is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile, methanol, etc.) ensure that the membrane material does not dissolve in the solvent. Contact the membrane manufacturer with solvent compatibility questions.

pH Range

The pH range for Atlantis™ columns is 2 to 8. Column lifetime will vary depending upon the temperature, the type and concentrationof buffer used. A listing of recommended and non-recommended buffers is given in Table 3. Please use this as a guideline whendeveloping methods.

Note: The use of phosphate buffer at pH 8 in combination with elevated temperatures will lead to shorter column lifetime.

Table 3: Buffer recommendations for using Atlantis™ columns from pH 2 to 8

Solvents

To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use.Pall Gelman Laboratory Acrodisc® filters are recommended. Solvents containing suspended particulate materials will generally clog the

Additive or Buffer range Used for Buffer pKa (±1 pH unit) Volatility Mass Spec? Comments

Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with Ammonium Formate salt. Used in 0.1-1.0% range.

Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with Ammonium Acetate salt. Used in 0.1-1.0% range.

Formate 3.75 2.75 – 4.75 Volatile Yes Used in the 1-10mM range. Note: sodium or(NH4COOH) potassium salts are not volatile.

Acetate 4.76 3.76 – 5.76 Volatile Yes Used in the 1-10mM range. Note: sodium or(NH4CH2COOH) potassium salts are not volatile.

Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.

Phosphate 2 7.2 6.20 – 8.20 Non-volatile No Above pH 7, reduce temperature / concentration and use guard column to maximize lifetime.


outside surface of the inlet distribution frit of the column. This will result in higher operating pressure and poorer performance.

Degas all solvents thoroughly before use to prevent bubble formation in the pump and detector. The use of an on-line degassing unit isalso recommended. This is especially important when running low pressure gradients since bubble formation can occur as a result ofaqueous and organic solvent mixing during the gradient.

Pressure

Atlantis™ columns can tolerate pressures of up to 6,000 psi (400 bar or 40 Mpa) although pressures greater than 4,000 – 5,000 psishould be avoided in order to maximize column and system lifetimes.

Temperature

Temperatures between 20˚C – 45˚C are recommended for operating Waters Atlantis™ columns in order to enhance selectivity, lower sol-vent viscosity and increase mass transfer rates. However, any temperature rise above ambient will have a negative effect on lifetimewhich will vary depending on the pH and buffer conditions used.

Scaling Up/Down Isocratic Methods

The following formulas will allow scale up or scale down, while maintaining the same linear velocity (retention time), and provide newsample loading values:

If column i.d. and length are altered: F2 = F1(r2/r1)2

or Load2 = Load1(r2/r1)2(L2/L1)

or Inj vol1 = Inj vol2 (r2/r1)2 (L2/L1)

Where: r = Radius of the column, in mm

F = Flow rate, in mL/min

L = Length of column, in mm

1 = Original, or reference column

2 = New column

Column Cleaning, Regenerating and Storage

Cleaning and Regeneration

A shift in retention or resolution may indicate contamination of the column. Flushing with a neat organic solvent is usually sufficient toremove the contaminant. If the flushing procedure does not solve the problem, purge the column with a sequence of progressively morenonpolar or hydrophobic solvents. For example, switch from water to tetrahydrofuran (THF) to methylene chloride. Return to the standardmobile phase conditions by reversing the sequence.

Guard columns need to be replaced at regular intervals as determined by sample contamination. When system backpressure steadilyincreases above a set pressure limit, it is usually an indication that the guard column should be replaced. A sudden appearance of splitpeaks is also indicative of a need to replace the guard column.

Storage

For periods longer than four days store the column in 100% acetonitrile. Do not store columns in buffered eluents. If the mobile phasecontained a buffer salt, flush the column with 10 column volumes of HPLC grade water (see Table 2 for common column volumes) andreplace with 100% acetonitrile for storage. Failure to perform this intermediate step could result in precipitation of the buffer salt in thecolumn when 100% acetonitrile is introduced. Completely seal column to avoid evaporation and drying out of the bed.

Note: If a column has been run with a mobile phase that contains formate (e.g., ammonium formate, formic acid, etc.) and is thenflushed with 100% acetonitrile, slightly longer equilibration times may be necessary when the column is re-installed and run again with aformate-containing mobile phase.

Troubleshooting

Changes in retention time, resolution, or backpressure are often due to column contamination. See the Column Cleaning,Regeneration and Storage section of this Care and Use Manual. Information on column troubleshooting problems may be found inHPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH, 1997) or the Waters HPLC Troubleshooting Guide(Literature code # 720000181EN).


I.D. Length Type 3 µm 5 µm

Atlantis™ dC18 Narrowbore Columns

1.0 mm 50 mm Column 186001279 186001281

1.0 mm 150 mm Column 186001283 186001285

2.1 mm 10 mm Guard 1860013771 1860013791

2.1 mm 20 mm Guard 1860013812 1860013832

2.1 mm 30 mm Column 186001287 186001289

2.1 mm 50 mm Column 186001291 186001293

2.1 mm 100 mm Column 186001295 186001297

2.1 mm 150 mm Column 186001299 186001301

Atlantis™ dC18 Analytical Columns

3.0 mm 50 mm Column 186001389 186001391

3.0 mm 100 mm Column 186001303 186001305

3.0 mm 150 mm Column 186001307 186001309

3.0 mm 250 mm Column — 186001311

3.9 mm 20 mm Guard 1860013133 1860013153

3.9 mm 50 mm Cartridge 1860013854 1860013874

3.9 mm 100 mm Column 186001393 186001395

3.9 mm 150 mm Column 186001317 186001319

4.6 mm 20 mm Guard 1860013213 1860013233

4.6 mm 30 mm Column 186001325 186001327

4.6 mm 50 mm Column 186001329 186001331

4.6 mm 75 mm Column 186001333 186001335

4.6 mm 100 mm Column 186001337 186001340

4.6 mm 150 mm Column 186001342 186001344

4.6 mm 250 mm Column — 186001346

1 – Requires Universal Sentry™ Guard Holder WAT0979582 – Requires Universal Sentry™ Guard Holder 1860002623 – Requires Universal Sentry™ Guard Holder WAT0469104 – Requires Cartridge End Fittings WAT037525

Ordering Information


Waters, Atlantis, Oasis, Alliance, Sentry and Sep-Pak are trademarks of Waters Corporation. Acrodisc is a registered trademark of Pall Gelman Laboratories.

Slipfree is a trademark of Keystone Scientific, Inc.©2002 Waters Corporation, 08/02 720000472EN

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