Symmetry ® Family of HPLC Columns · Symmetry® columns demonstrate superior reproducibility over...

Symmetry® Familyof HPLC Columns

Symmetry® • SymmetryShield™ • Symmetry300™ • SymmetryPrep™

Solutions for Common HPLC Column Problems

Physical Characteristics

Packing Chemistry Particle Particle Pore Carbon End-Size Shape Size Load capped

Symmetry® C18 3.5, 5 µm Spherical 100Å 19% YesC8 3.5, 5 µm Spherical 100Å 12% Yes

SymmetryPrep™ C18 7 µm Spherical 100Å 19% YesC8 7 µm Spherical 100Å 12% Yes

SymmetryShield™ RP8 3.5, 5, 7 µm Spherical 100Å 15% YesRP18 3.5, 5, 7 µm Spherical 100Å 17% Yes

Symmetry300™ C18 3.5, 5 µm Spherical 300Å 8.5% YesC4 3.5, 5 µm Spherical 300Å 2.8% Yes

Symmetry® Columns—the solution for today’sdemanding analytical requirements

Column-to-column and batch-to-batch repro-ducibility is not consistent

Severe or excessive peak tailing

Column selectivity not resolvingcritical peaks

Difficulty resolving individual compounds incomplex peptide or protein samples

Scaling-up analytical method to prepara-tive scale requires too many adjustments toachieve good isolation and purification

• Increased labor costs due to individualcolumn QC testing

• Revalidate/redevelop method with eachnew batch of columns

• Increased number of experiments tooptimize analytical method

• Increased number of experiments requiredto optimize method

• Increased cost and time to isolatecompounds

• Higher costs due to method developmenttime and labor

• Higher cost of equipment, samples andprep columns

• Symmetry® brand of HPLC columns have the tightest specifi-cations in the market to provide the most consistent results

• Symmetry® columns achieve outstanding year-to-year repro-ducibility to give long life to established analytical methods

• SymmetryShield™ columns with embedded polartechnology provide less peak tailing for faster andeasier method development

• SymmetryShield™ columns are the perfect complement toSymmetry® columns, providing different selectivity andenabling column selection to maximize resolution

• Symmetry300™ columns demonstrate the highest peakcapacity of all leading brands

• Excellent reproducibility for separating large numbers ofpeptides or proteins

• SymmetryPrep™ columns save development time with directscale-up while maintaining resolution

Problem Impact Solution and Benefit

Column: Symmetry® C18,4.6 x 150 mm, 5 µm

Mobile Phase A: H2OMobile Phase B: MeCNMobile Phase C: pH 3.75; 100 mM

NH4COOH in H2OFlow Rate: 1.4 mL/minIsocratic: 30% A; 60% B; 10% CInjection Volume: 5.0 µLTemperature: 30 °CDetection: UV @ 233 nm

Instrumentation: Waters Alliance® HT 2795Waters 996 PDA detectorSampling Rate: 5 pts./sec

Symmetry® Columns

Setting the Standard for Reproducibility

As today’s chemists establish new analyti-cal methods for the latest pharmaceuticaland biopharmaceutical products, the selec-tion of a reproducible HPLC column isessential. The selected column needs toprovide the same chromatographic resultsover the life of the new drug product.Symmetry® columns demonstrate superiorreproducibility over many years. Batchesrandomly selected over the past 5 yearsdemonstrate excellent reproducibility in theexample shown.

Reproducibility is our number one priority insupplying Symmetry® columns. This excel-lent reproducibility is a result of our com-mitment to maintaining the tightest specifi-cations in the HPLC column industry.Symmetry® columns are engineered withhigh purity raw materials, tightly controlledmanufacturing processes and column pack-ing procedures that provide today’s scien-tists with the best, most reproducible HPLCcolumn available.

Our goal is to supply a family of HPLCcolumns that you can rely on for ruggedand robust methods. Symmetry® columnslet you increase your laboratorys' produc-tivity, and allow easier method transferbetween labs and around the globe.

A three-page Certificate of Analysis isincluded with every Symmetry® column.We report both our specifications,which are the tightest in the industry,and the results of the 22 critical testseach column must pass before it carriesthe Symmetry® brand.

0.00

1 2

3

4

1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50 15.00

RSD’s for Retention Times

1. Terbinafine HCl 0.7%

2. Ibuprofen 0.8%

3. Lovastatin 0.6%

4. Simvastatin 0.7%

Batch 136 (1998)

Batch 142 (1998)

Batch 149 (1999)

Batch 151 (2000)

Batch 156 (2001)

Unmatched Year-to-year Reproducibility

Comprehensive Certificate of Analysis

1

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The Benefit of NarrowSpecification Ranges

Symmetry® columns have the narrowest ligand surface coverage specifications inthe industry. Surface coverage (ligand density) is one of the most importantparameters affecting reproducibility. Theimpact of a small variation in surface cov-erage on the amitriptyline/acenaphthenealpha specification for Symmetry® C18

columns is shown here.

Narrow column specifications benefitthe chromatographer in achieving repro-ducible results. The surface coverage re-quirements of Symmetry® are the tightestspecifications in the industry, resulting inminimal shifts in resolution and more repro-ducible batch-to-batch and column-to-column results.

Long Lifetime

Columns that last longer save you money.Symmetry® columns can last over 10,000injections with minimal loss in efficiency,minimal increase in backpressure andminimal change in retention time.

Symmetry® columns deliver guaranteedconsistent performance optimizing thetwo key factors that control column lifetimewhich are hydrolytic stability of the pack-ing material and mechanical stability ofthe packed bed.

1.5 2 2.5 3 3.5 40.8

1

1.2

1.4

1.6

1.8

2

0.814.70

5.37

2.864.14

0

1.5 2 2.5 3 3.5 4

2.75

2.6

3.09 3.31

3.75

Narrow Specification Ranges Provide Smaller Shifts in Alpha (α)

Smaller Shifts in Alpha (α) ReduceChanges in Peak Resolution

C18 Ligand Surface Coverage Specification Ranges

Specification Rangesfor Symmetry® C18

Minimal Changes in Resolution Means More Reproducible Results*

* Based on vendors column product specifications for ligand density

Outstanding Packed Bed Stability—10,000 InjectionsColumn: Symmetry® C8 3.9 mm x 150 mm, 5 µm

with Sentry™ Guard Column3.9 mm x 20 mm

Mobile Phase: Water/methanol/glacial acetic acid 79:20:1

Flow Rate: 1.0 mL/minuteInjection Volume: 10 µLTemperature: 25°CDetector: UV @ 254Analytes: 1. Sulfanilamide

2. Sulfadiazine3. Sulfathiazole4. Sulfamerazine5. Sulfamethazine6. SuccinylsulfathiazoleInjection 10,000

Start

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1

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45

6

The long-term mechanical stability of Symmetry® columns is demonstrated in this analysis of sulfa drugs. Over 10,000 injections were runwith minimal loss in column efficiency. The use of the Sentry™ guard column is critical to achieving these types of column lifetimes.

Specification X

Specification Y

Symmetry® C18

Symmetry® Columns

Symmetry® C18

Column X

Column Y

C18 Surface Coverage (µmol/m2)

C18 Surface Coverage (µmol/m2)

Alp

ha fo

r Am

itrip

tylin

e/A

cena

phth

ene

no upper limit

Maximum Resolution Minimum Resolution

2

R=N4( ) α-1

α( ) K2

K2+1( )R = resolution

N = number of peaksα = alpha valuek = peak retention

Superior Peak ShapeRegardless of Mobile Phase pH

The performance of any reversed-phasecolumn is determined by two factors.

First, the bonded phase coverage(hydrophobicity); and second, the activityof the unbonded, residual silanols on the silica surface.

Symmetry® columns deliver superior peakshape across the pH range of 2–8. This is due to the high bonding density and minimal residual silanol activity of the packing materials.

Now you can work at neutral pH or lowpH to optimize your method without compromising peak shape.

Speed and Resolution

The speed you need for high throughputlead generation – speed that enablesrapid analysis and purification of thousands of samples per day anddecreases run times by 50% withoutsacrificing resolution.

+ Achieve increased resolution withsmaller particle size material in thesame gradient time.

+ Increase throughput and resolutionwith smaller particle size if flow rateis increased

+ Reduce analysis time by 50% byusing shorter columns

+ Maintain resolution by scalinggradient volume proportionally tocolumn volume

The Influence of Mobile Phase pH on Peak Shape of Amitriptyline

Symmetry® columns deliver superior peak shape forbasic compounds across a wide pH range.

Amitriptyline Tailing Versus Buffer pH

Conventional C18

Symmetry® C18 Column

Buffer pH

USP

Tai

ling

Fact

or

Increased Speed While Maintaining Resolution

Columns: See chromatogramMobile Phase A: 0.1% TFA in waterMobile Phase B: 0.1% TFA in acetonitrileFlow Rate: 1.0 mL/minGradient*: Time Profile

(min) %A %B0.00 100 04.00 40 60

Injection Volume: 1 µLTemperature: 30.0 °CDetection: UV @ 254 nm

* same gradient applies for 1, 2 and3 minutes as specified.

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

Symmetry® C18, 3.5 µm, 2.1 mm x 10 mmGradient time = 1 minute

Symmetry® C18, 3.5 µm, 2.1 mm x 20 mmGradient time = 2 minutes



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AU

SymmetryShield™ HPLC columns have setthe standard of performance for peakshape of basic compounds. This reversed-phase column is based on Waters’ patent-ed embedded polar group technology thatliterally “shields” the silica’s residual surfacesilanols from highly basic analytes.

Shield Technology

The embedded polar group, which is inclose proximity to the silica surface, furtherreduces the activity of the surface silanols inSymmetryShield™ packing materials.

As a result, SymmetryShield™ columns:

+ Deliver significantly improved peakshape and resolution over a broadpH range

+ Exhibit less retention of basic com-pounds than conventional C18 andC8 columns

In fact, SymmetryShield™ columns deliverUSP Tailing Factors of less than 1.2 overa pH range between pH 2 and pH 8.By delivering lower limits of detectionand quantitation, SymmetryShield™

columns will enable you to develop newmethods faster and improve the produc-tivity of your laboratory.

SymmetryShield™ Columns

Raising the Standard for Superior Peak Shape

Si

CH3 CH3

O PolarGroup

Reduce interaction with silanolsImprove water-wettability

Industry Leader for Peak Shape of Basic Compounds

Column: a. SymmetryShield™ RP18 3.9 x 150 mmb. Leading Brand C18

Mobile Phase: 65% methanol; 35% 20 mM KH2PO4/K2HPO4 @ pH 7

Flow Rate: 1.0 mL/minuteDetection: UV @ 254 nmTemperature: 23°CAnalytes: 1. Uracil

2. Propranolol3. Butylparaben4. Dipropylphthalate5. Naphthalene6. Amitriptyline7. Acenaphthene

a: SymmetryShield™ RP18

b: Leading Brand C18

In this comparison test, SymmetryShield™ RP18has the lowest USP tailing factor for the basicprobe amitriptyline under the most demandingpH 7 mobile phase conditions.

USP Tailing Factor

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USP Tailing Factor = 1.2

SymmetryShield™ RP18

Brand L

Brand HP

Brand HE

Brand DO

Brand DA

Brand I

Brand P

Brand E

1.2

1.5

1.66

1.75

1.78

1.9

1.95

2.39

3.6

0 1 2 3 4

4

USP Tailing Factor = 1.9

SymmetryShield™ Columns Deliver Unique Selectivity

Leading Brand C8

3.9 x 150 mm column,straight chain alkyl

SymmetryShield™ RP8

3.9 x 150 mm column,embedded polar ligand

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Unique Selectivity

Obtaining a good separation is para-mount. SymmetryShield™ columns exhibitless retention for basic compounds due tolower silanol activity than conventionalC18 and C8 columns. SymmetryShield™

columns are an excellent methods devel-opment tool.

In the example shown, a leading phar-maceutical company was faced with achallenging separation. It was onlywhen a SymmetryShield™ column wassubstituted for a conventional C8 columnthat a much better separation wasachieved without having to changemobile phase conditions.

Perfect for Use with HighlyAqueous Mobile Phases

SymmetryShield™ columns with ShieldTechnology are the perfect choice for appli-cations that require low organic, highlyaqueous mobile phase conditions. Thesereversed-phase particles are water-wettableallowing better interaction between the par-ticles and your sample. The result is stableretention in highly aqueous mobile phaseswith superior peak shape.

Most HPLC columns lose their retention whenrunning low or 0% organic mobile phases if the flow is stopped. When this happens thebonded particle pores become de-wetted. The result is termed “pore dewetting” and youlose the sensitivity of your analyte.

SymmetryShield™ columns prevent this fromhappening because the embedded polargroup maintains the wetted pore surface, ensuring retention of your analyte and eliminating the need for a lengthy re-wettingand column re-equilibration process.

Standard Silica C8 Columns — Pore Dewetting

SymmetryShield™ RP8 Columns — No Pore Dewetting!

Column: Standard C8 Silica Column4.6 mm x 150 mm, 5 µm

0 2 4 6 8 10

Mobile Phase: Aqueous0.1% Acetic Acid

After pump stoppedand re-started(Pore dewetting: ~ 100%)

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InitialAmoxicillin

No retention for analyte

Column: SymmetryShield™ RP8

4.6 mm x 150 mm, 5 µm

0 2 4 6 8 10

Mobile Phase: Aqueous0.1% Acetic Acid

After pump stoppedand re-started(Pore dewetting: ~ 3%)

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Initial

Amoxicillin

No change inretention time

5

Symmetry300™ Columns

High Recoveries of Proteins and Peptides

Symmetry300™ columns are wide-pore,reversed-phase columns which are built on the Symmetry® column platform, thestandard that the pharmaceutical industryhas come to rely on for sensitive, ruggedand robust HPLC analyses.

Symmetry300™ C18 columns provide better peak shape and peak capacity compared to other leading brands whenseparating biologically active peptides. Inthe examples shown, Symmetry300™

columns demonstrate superior perform-ance when simultaneously separatinghydrophilic and hydrophobic peptides,as well as neutral and highly basic peptides. Also, hydrophobicity (Meekretention prediction1) is included. Thelower hydrophobicity values indicate the peptide is more hydrophilic.

Symmetry300™ columns perform better at high and low mass load conditions.For mass spectrometry applications, we recommend low TFA mobile phases or0.1% formic acid.

Column-after-column, no matter who usesyour methods or where they are usedaround the world, the Symmetry300™

columns are the best solution for validationcompliance.

Symmetry300™ columns are available intwo particle sizes: 3.5 µm and 5 µm, andin two chemistries: C4 for large peptidesand proteins, and C18 for smaller peptides.

Peptide Separation at High Mass Load (0.1% TFA)

Peptide Separation at Low Mass Load (0.1% Formic Acid)

Injection Volume:Temperature: 40°CDetection: UV 220nmInstrument: Alliance® 2690

Columns: 4.6 x 50 mm, 5 µmMobile Phase A: 0.1% TFA in H2OMobile Phase B: 0.08% TFA in ACNFlow Rate: 0.75 ml/minGradient: 0-50% B in 25 min.

Injection Volume:Temperature: 40°CDetection: UV 220nmInstrument: Alliance® 2690

Columns: 4.6 x 50 mm, 5 µmMobile Phase A: 0.1% formic acid in H2OMobile Phase B: 0.08% formic acid in ACNFlow Rate: 0.75 ml/minGradient: 0-50% B in 25 min.

Name MW pI Hydrophobicity Number of(Meek) Amino Acids

1. L2275 986.6 12.04 2.8 92. A6677 1001.5 9.34 3.0 103. Bradykinin 1060.2 12.00 42.6 94. Angiotensin II 1046.2 7.35 43.7 85. Angiotensin I 1296.5 7.51 54.0 106. Substance P 1347.6 11.20 52.7 117. Renin substrate 1759.0 7.61 63.5 148. Insulin B chain 3495.9 7.51 129.4 309. Melittin 2845.8 12.06 92.3 26

1 J. L. Meek and Z. L. Rossetti, J. Chromatogr., 211 (1981) 15-28.pI = Isoelectric pH

* Note:

Peak Capacity (P) is defined as thenumber of theoretical peaks that canfit into a chromatographic run within acertain gradient time

P = 1 + (tg/w5σ)

Where tg = gradient time

w5σ = peak measured at 4.4% of peak height

Biologically Active Peptide Mixture1

1 2

3 4 5 67 8

9

250 5 10 15 20

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Leading 300Å C18 Brand 4.6 x 50 mm columnP = 70.0*

Symmetry300™ C18 4.6 x 50 mm columnP = 98.8*

3 4 7 5

6 8

9

2

0 255 10 15 20

Leading 300Å C18 Brand 4.6 x 50 mm columnP = 62.3*

Symmetry300™ C18 4.6 x 50 mm columnP = 93.1*

6

0.00 2.00 4.00 6.004.00 4.00 6.00 6.00

Cytochrome c Tryptic Map Q.C. TestWaters batch release protocol includes a tryptic mapof cytochrome c which is used to test for reproducibili-ty of retention time and resolution. In this test, the tryp-tic map was run on four Symmetry300™ columns,each containing a different batch of packing.

Unsurpassed Reproducibility

Column: Symmetry300™ C18 3.9 x 150 mm, 5 µmMobile Phase A: 0.05% TFA in waterMobile Phase B: 0.1% TFA in acetonitrileFlow rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.00 100 045 72 28

Injection volume: 20 µLTemperature: 35 °CDetection: UV @ 220 nm

Sharpest Peaks for HighestResolution

The heart of the column is the high puritybase deactivated silica. The silica used inSymmetry300™ columns is synthesizedusing ultrapure organic reagents, resultingin high purity with very low silanol activity.

In addition, when combined with the highsurface coverage of the bonded phasethe result is higher recoveries of proteinsand peptides even when injected at low concentrations.

Symmetry300™ columns deliver thesharpest peaks for biomolecules anddeliver more peak capacity for complextryptic maps.

They are packed to the highest efficiencyand reproducibility of any product on themarket. This results in improved precisionfor impurity and stability assays, more accu-rate quantitation, and adaptability to morerugged methods.

Greater Peak CapacityTryptic Map of Bovine Serum Albumin

Injection Volume: 20 µLTemperature: 35 °CDetection: UV @ 214 nm

Column: Symmetry300™ C18, 4.6 mm x 50 mmMobile Phase: Eluent A: 0.1% TFA in water

Eluent B: 0.1% TFA in acetonitrileFlow Rate: 1.0 mL/minGradient: Time Profile

(min) %A %B0.00 100 045 70 30

Protein: Symmetry300™ C4 Versus Competitors

Columns: 4.6 x 50 mm, 5 µmMobile Phase A: 0.1% TFA in waterMobile Phase B: 0.08% TFA in acetonitrileFlow Rate: 0.75 mL/minGradient: Time Profile

(min) %A %B0.00 80 2015.00 35 65

Injection Volume: 20 µLTemperature: 40° CDetection: UV @ 220 nmInstrument: Alliance® 2790, 996 PDAAnalytes: 1. RNAse

2. BSA3. B-Lactoglobulin4. Ovalbumin

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Symmetry300™

C4 columnP = 138.56

Competitive 300ÅC18 columnP = 90.09

Excellent Protein Separations

Symmetry300™ C4 columns have the high-est average peak capacity when com-pared to over 20 leading brands of HPLCcolumns. In the example shown, which isconsidered to be a difficult protein separa-tion, the Symmetry300™ C4 column pro-vided a better separation with better peakshape compared to a leading competitive300Å column.

Scale-up CapabilityColumn: See chromatogramMobile Phase A: 0.1% TFA in waterMobile Phase B: 0.08% TFA in acetonitrileFlow Rate: A: 15 mL/min

B: 1.0 mL/minGradient*: Time Profile

(min) %A %B0.00 100 04.00 40 60

Injection Volume: A: 100 µLB: 5 µL

Temperature: 30 °CDetection: UV @ 254 nmAnalytes: 1. 1-hydroxy-7-aza-benzotriazole

2. 4-methylbenzene sulfonamide3. methyl 3-amino-2-thiophenecarboxylate4. 4-aminobenzophenone

SymmetryPrep™ Columns

Fast, Direct Scale Up From Analytical to Prep

Direct scale-up from analytical to prepara-tive applications – columns that deliver therequisite capacity to take new drug sub-stances from the lead generation to thelead optimization stage of the drug discov-ery process. The high capacity ofSymmetry® columns enables you to scale-up using the same analytical method inorder to produce 10-100 milligrams of anew drug substance to expedite the start ofpreclinical drug metabolism studies.

If you’re involved with purifying new leadcandidates, supporting toxicology workor isolating impurities and degradants,there is a Symmetry® column to meet yourneeds. SymmetryPrep™ 7 µm columns havechromatographic selectivity identical toSymmetry® 3.5 µm and 5 µm columns. AnHPLC method developed on a Symmetry®

analytical column can be scaled up direct-ly to a SymmetryPrep™ column with no fur-ther method development. SymmetryPrep™

columns deliver sharp, symmetrical peaksusing simple, volatile mobile phase bufferssuch as TFA and ammonium acetate.

Should your preparative separationsrequire the efficiency of a 5 µm column,an extensive line of Symmetry® semiprepcolumns packed with 5 µm particles areavailable.

0

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0 4.00

0

1

AU

AU

1

1

2

2

3

3

4

a

b

4

Symmetry® C18

19 mm x 50 mm, 5 µm

Symmetry® C18

4.6 x 50 mm, 5 µm

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High CapacityColumn: Symmetry® C18, 5 µm, 19 mm x 50 mmMobile Phase A: 0.1% TFA in waterMobile Phase B: 0.1% TFA in acetonitrileFlow Rate: 15 mL/minGradient: 0-60% B in 4 minutesTemperature: 30 °CDetection: UV @ 254 nm

0.80

AU

0.60

0.40

0.20

4.00

3.00

2.00

1.00

0.00

4.00

3.00

2.00

1.00

0.00

0.00 2.00 4.00

0.00 2.00 4.00

0.00 2.00 4.00

Injection volume: 100 µL

Injection volume: 1,000 µL

Injection volume: 2,000 µL

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10x

20x

Impurity Profiling: ResolutionMaintained when Scaling-up

SymmetryPrep™ columns are manufacturedunder the same rigid standards asSymmetry® analytical columns whichallows for direct scale-up while maintainingresolution as shown. This can be a significant benefit in saving both time andlabor costs when isolating and purifyingtrace analytes.

Extended Column Lifetimes

SymmetryPrep™ columns achieve longer column lifetimes with minimal change of efficiency, backpressure, or retention.

Symmetry® Guard ColumnsRecommended

SymmetryPrep™ columns lifetimes can be further extended with negligible increase in run time with the addition of aSymmetry® guard column. The guard column will protect the prep column andcan be replaced as necessary.

The Symmetry® guard column can protectthe prep column against pressure shock.Pressure shock associated with the samplervalve switching (particularly with large injec-tion volumes of different viscosity sample solvents) can cause packed beddamage especially if the surface area islarge (i.e. 19 mm i.d. column or greater).

Valerophenone Impurities: Scaling up fromSymmetry® 5 µm to SymmetryPrep™ 7 µm Columns

Column: a: Symmetry® C18, 3.9 mm x 150 mm, 5 µm b: SymmetryPrep™ C18, 7.8 mm x 300 mm, 7 µm

Mobile Phase: 60% acetonitrile/40% waterFlow Rate: a: 0.70 mL/min

b: 5.60 mL/minSample: 4 mg/mL valerophenoneInjection Volume: a: 100 µL

b: 800 µLDetection: UV @ 340 nm

a

b

Long Column Lifetimes

Column: Symmetry® C18, 19 x 50 mm, 5 µmSymmetry® Guard C18, 7.8 x 10 mm, 5 µm

Mobile Phase A: 0.1% TFA in waterMobile Phase B: 0.1% TFA in acetonitrileFlow Rate: 12 mL/minGradient: 0-60% B in 4 minutesInjection Volume: 100 µLTemperature: 30 °CDetection: UV @ 254 nm

Extend column lifetime withnegligible increase in run time

0.00 2.00 4.00 6.00

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Injection #1

Injection #1045(before Guard Columnwas changed)

Injection #1120(after Guard Columnwas changed)

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NEW—Purification andIsolation Cartridge Holders

Symmetry® Capillary Columns

Columns and Chemistries: Small in Size, Big in Performance

When it comes to LC/MS applications, thechoice of column can greatly impact speedand sensitivity gains. With this in mind,Waters has developed microbore and cap-illary columns to meet your LC/MS methoddevelopment needs.

Symmetry® columns provide the higheststandard of reproducibility with unmatchedpeak symmetry for maximum sensitivity andaccurate quantification.

Method Development

Stability-Indicating HPLC Method Development Kit

Waters offers a four column MethodDevelopment Kit with a step-by-step pro-tocol for selecting HPLC columns andidentifying optimum pH levels. TheMethod Development Kit also includes acomprehensive Applications Notebook.

Symmetry® C18 3.5 µm column 4.6 x 100 mm

SymmetryShield™ RP18 3.5 µm column 4.6 x 100 mm

XTerra® MS C18 3.5 µm column 4.6 x 100 mm

XTerra® RP18 3.5 µm column 4.6 x 100 mm

Comprehensive Applications Notebook.

Part Number :186001100

Applications Notebook with Proven Methodology

Step-by-step four-column approach completed at pH 3, pH 7, and pH 10on the following drugs:

• Diltiazem Hydrochloride• Fluconazole• Lansoprazole• Paclitaxel• Pravastatin Sodium• Risperidone• Simvastatin• Terbinafine Hydrochloride• Zidovudine

Polyimide coating Bare fused silica Frit

TipPacked bed length, X

Fused-silica tubing, ≈ 50 cm360 µm OD x 75 µm ID

Polyimide coating

Bare fused silica

Frit

Packed bed

Fused-silica tubing, 50 cm360 µm OD x 75 µm

Distal endPolished edge

10

PicoFrit™ 75 µm Columns

IntegraFrit™ 75 µm Columns

PicoFrit™ columns allow you to do femtomole peptide analysis. You can also spray directly from column to inletof a mass spectrometer and minimize post-column loss of sample.

IntegraFrit™ columns provide very low back pressure and are free of adhesives, binders and polymers. They canbe used with both ESI-MS and MALDI, and are Biomolecule and MS-compatible.

http://www.waters.com/stability-indicating

Particle Part No. Part No.Dimension Size C18 C8

1.0 x 50 mm 3.5 µm WAT106056 WAT1060521.0 x 150 mm 3.5 µm WAT248059 WAT2480722.1 x 30 mm 3.5 µm WAT058973 WAT0589772.1 x 50 mm 3.5 µm WAT200650 WAT2006242.1 x 100 mm 3.5 µm WAT058965 WAT0589612.1 x 150 mm 3.5 µm WAT106005 WAT1060113.0 x 100 mm 3.5 µm 186000696 1860006983.0 x 150 mm 3.5 µm 186000695 1860006974.6 x 30 mm 3.5 µm 186000271 1860002704.6 x 50 mm 3.5 µm WAT200625 WAT2006204.6 x 75 mm 3.5 µm WAT066224 WAT0662004.6 x 100 mm 3.5 µm WAT066220 WAT0662044.6 x 150 mm 3.5 µm WAT200632 WAT200630

2.1 x 50 mm 5 µm 186000206 1860002122.1 x 150 mm 5 µm WAT056975 WAT0569553.0 x 150 mm 5 µm WAT054200 WAT0542303.0 x 250 mm 5 µm 186000690 1860006913.9 x 150 mm 5 µm WAT046980 WAT0469704.6 x 50 mm 5 µm 186000207 1860002134.6 x 150 mm 5 µm WAT045905 WAT0459954.6 x 250 mm 5 µm WAT054275 WAT054270

Symmetry® Analytical Columns


2.1 x 20 mm 3.5 µm 186000269 1860002682.1 x 50 mm 3.5 µm 186000152 1860001492.1 x 100 mm 3.5 µm 186000151 1860001532.1 x 150 mm 3.5 µm 186000150 1860001484.6 x 75 mm 3.5 µm WAT066260 WAT0662104.6 x 100 mm 3.5 µm WAT066265 WAT066215

3.9 x 50 mm 5 µm WAT054220 WAT0542403.9 x 150 mm 5 µm WAT054205 WAT0542354.6 x 150 mm 5 µm WAT054210 WAT0542554.6 x 250 mm 5 µm WAT054215 WAT054245

Symmetry® Cartridge Columns (Requires endfittings, see Page 13)


2.1 x 10 mm 3.5 µm WAT106127 WAT1061283.9 x 20 mm 5 µm WAT054225 WAT054250

Symmetry® Sentry™ Guard Columns (2 per pack)(Requires Sentry™ Guard Holders, see Page 13)


0.32 x 50 mm 3.5 µm 186000945 1860009460.32 x 100 mm 3.5 µm 186000951 1860009520.32 x 150 mm 3.5 µm 186000957 186000958

0.32 x 50 mm 5 µm 186000929 1860009300.32 x 100 mm 5 µm 186000935 1860009360.32 x 150 mm 5 µm 186000347 186000941

Symmetry® Capillary Columns

Symmetry® Columnsand Cartridges Dimension Particle Size Part Number

75 µm x 50 mm 3.5 µm 18600140775 µm x 100 mm 3.5 µm 18600140875 µm x 100 mm 5 µm 18600140975 µm x 150 mm 5 µm 186001410

Symmetry® C18 PicoFrit™ Columns

Dimension Particle Size Part Number

75 µm x 50 mm 3.5µm 18600140375 µm x 100 mm 3.5 µm 18600140475 µm x 100 mm 5 µm 18600140575 µm x 150 mm 5µm 186001406

Symmetry® C18 IntegraFrit™ Columns

Particle Part No. Part No.Dimension Type Size C18 C8

4.6 x 150 mm Column 3.5 µm WAT094240 WAT0942372.1 x 150 mm Column 5 µm WAT094234 WAT0942313.0 x 150 mm Column 5 µm WAT054446 WAT0544343.9 x 150 mm Column 5 µm WAT047210 WAT0469554.6 x 150 mm Column 5 µm WAT054448 WAT0544354.6 x 250 mm Column 5 µm WAT054450 WAT054438

3.9 x 150 mm Cartridge* 5 µm WAT054452 WAT0544404.6 x 150 mm Cartridge* 5 µm WAT054454 WAT0544424.6 x 250 mm Cartridge* 5 µm WAT054456 WAT054444

* Require Endfittings, see Page 13

Symmetry® Column and Cartridge ColumnMethod Validation Kits Three Columns from Three Different Batches to Test Reproducibility.


7.8 x 10 mm 5 µm 186000711 1860007127.8 x 50 mm 5 µm 186000208 1860002147.8 x 100 mm 5 µm 186000209 18600021519 x 10 mm 5 µm 186000715 18600071619 x 50 mm 5 µm 186000210 18600021619 x 100 mm 5 µm 186000211 18600022930 x 50 mm 5 µm 186000235 18600023730 x 100 mm 5 µm 186000236 186000238

7.8 x 10 mm 7 µm 186000713 1860007147.8 x 150 mm 7 µm WAT066288 WAT0662857.8 x 300 mm 7 µm WAT066235 WAT06622519 x 10 mm 7 µm 186000717 18600071819 x 150 mm 7 µm WAT066240 WAT06622819 x 300 mm 7 µm WAT066245 WAT066230

Purification and Isolation Cartridge Column Holders

7.8 x 10 mm 5, 7 µm — 18600070819 x 10 mm 5, 7 µm — 186000709

Replacement o-ring 7.8 mm, 2 pkg 700001019Replacement o-ring 19 mm, 2 pkg 700001020

SymmetryPrep™ Columns for Purification and Isolation

11

SymmetryShield™

Columns and Cartridges

Symmetry300™

Columns and Cartridges

Particle Part No. Part No.Dimension Size RP18 RP8

1.0 x 50 mm 3.5 µm 186000175 WAT1060601.0 x 150 mm 3.5 µm 186000176 WAT1060482.1 x 30 mm 3.5 µm 186000171 WAT1060012.1 x 50 mm 3.5 µm 186000172 WAT0942572.1 x 100 mm 3.5 µm 186000173 WAT0589692.1 x 150 mm 3.5 µm 186000174 WAT1060083.0 x 100 mm 3.5 µm 186000700 1860007033.0 x 150 mm 3.5 µm 186000699 1860007024.6 x 50 mm 3.5 µm 186000177 WAT0942604.6 x 75 mm 3.5 µm 186000178 WAT0942634.6 x 100 mm 3.5 µm 186000179 WAT0942664.6 x 150 mm 3.5 µm 186000180 WAT094269

2.1 x 50 mm 5 µm 186000217 1860002232.1 x 150 mm 5 µm 186000111 WAT0942453.0 x 150 mm 5 µm 186000692 WAT0942433.0 x 250 mm 5 µm 186000693 1860006943.9 x 150 mm 5 µm 186000108 WAT2006554.6 x 50 mm 5 µm 186000218 1860002244.6 x 150 mm 5 µm 186000109 WAT2006624.6 x 250 mm 5 µm 186000112 WAT200670

SymmetryShield™ Columns


2.1 x 50 mm 3.5 µm 186000168 1860001472.1 x 100 mm 3.5 µm 186000167 1860001462.1 x 150 mm 3.5 µm 186000166 1860001454.6 x 75 mm 3.5 µm 186000183 WAT0942724.6 x 100 mm 3.5 µm 186000170 WAT094275

3.9 x 50 mm 5 µm — WAT0942483.9 x 150 mm 5 µm 186000106 WAT2006584.6 x 150 mm 5 µm 186000110 WAT2006654.6 x 250 mm 5 µm 186000113 WAT200661

SymmetryShield™ Cartridge Columns (Requires endfittings, see Page 13)


2.1 x 10 mm 3.5 µm 186000169 WAT1061293.9 x 20 mm 3.5 µm 186000701 1860007043.9 x 20 mm 5 µm 186000107 WAT200675

SymmetryShield™ Sentry™ Guard Columns (2 per pack)(Requires Sentry™ Guard Holders, see Page 13)


19 x 10 mm 5 µm 186001835 18600184119 x 50 mm 5 µm 186001836 18600184219 x 100 mm 5 µm 186001837 18600184319 x 150 mm 5 µm 186001838 186001844

19 x 150 mm 7 µm 186001839 18600184519 x 300 mm 7 µm 186001840 186001846


7.8 x 10 mm 5, 7 µm — 18600070819 x 10 mm 5, 7 µm — 186000709


SymmetryShield™ Columns for Purification and Isolation


1.0 x 150 mm 3.5 µm 186000185 1860002762.1 x 50 mm 3.5 µm 186000187 1860002772.1 x 100 mm 3.5 µm 186000188 1860002782.1 x 150 mm 3.5 µm 186000200 1860002794.6 x 50 mm 3.5 µm 186000201 1860002804.6 x 75 mm 3.5 µm 186000189 1860002814.6 x 100 mm 3.5 µm 186000190 1860002824.6 x 150 mm 3.5 µm 186000197 186000283

2.1 x 150 mm 5 µm WAT106172 1860002853.9 x 150 mm 5 µm WAT106154 1860002864.6 x 50 mm 5 µm WAT106209 1860002874.6 x 150 mm 5 µm WAT106157 1860002884.6 x 250 mm 5 µm WAT106151 186000289

Symmetry300™ Analytical Columns




SymmetryShield™ Capillary Columns

Particle Part No. Part No.Dimension Type Size RP18 RP8

2.1 x 150 mm Column 3.5 µm 186000182 —4.6 x 150 mm Column 3.5 µm 186000181 WAT0942782.1 x 150 mm Column 5 µm 186000100 WAT0942543.0 x 150 mm Column 5 µm - WAT0942513.9 x 150 mm Column 5 µm 186000104 WAT2105944.6 x 150 mm Column 5 µm 186000103 WAT2105884.6 x 250 mm Column 5 µm 186000102 WAT210591

3.9 x 150 mm Cartridge* 5 µm 186000105 WAT2105824.6 x 150 mm Cartridge* 5 µm 186000101 WAT2105854.6 x 250 mm Cartridge* 5 µm 186000114 WAT210579

* Require endfittings, see Page 13

SymmetryShield™ Column and Cartridge ColumnMethod Validation Kits Three Columns from Three Different Batches to Test Reproducibility.

12

Cartridge and Guard Columns

Integrated Sentry™ Guard Holder to gowith cartridge end-fittings (see above)3.0 x 20 mm, 3.9 x 20 mm – use Part Number WAT046905

Universal Sentry™ Guard Holder (Use on any column)

2.1 x 10 mm use Part Number WAT0979582.1 x 20 mm use Part Number 1860002623.0 x 20 mm use Part Number WAT0469103.9 x 20 mm use Part Number WAT0469104.6 x 20 mm use Part Number WAT046910

Particle Part No.Dimension Size C18

19 x 10 mm 5 µm 18600184719 x 50 mm 5 µm 18600184819 x 100 mm 5 µm 18600184919 x 150 mm 5 µm 186001850


7.8 x 10 mm 18600070819 x 10 mm 186000709


Symmetry300™ Columns for Purification and Isolation

Particle Part No.Dimension Size C18

2.1 x 50 mm 3.5 µm 1860001992.1 x 100 mm 3.5 µm 1860001912.1 x 150 mm 3.5 µm 1860001964.6 x 75 mm 3.5 µm 1860001924.6 x 100 mm 3.5 µm 186000193

3.9 x 150 mm 5 µm WAT1061694.6 x 150 mm 5 µm WAT1061634.6 x 250 mm 5 µm WAT106160

Symmetry300™ Cartridge Columns (Requires endfittings, see below)


2.1 x 10 mm 3.5 µm 186000198 1860002753.9 x 20 mm 5 µm WAT106166 186000284

Symmetry300™ Sentry™ Guard Columns (2 per pack)(Requires Sentry™ Guard Holders, see below)




Symmetry300™ Capillary Columns

Particle Part No. Part No.Dimension Type Size C18 C4

2.1 x 150 mm Column 3.5 µm 186000194 1860002904.6 x 150 mm Column 3.5 µm 186000195 186000291

2.1 x 150 mm Column 5 µm WAT106193 1860002923.9 x 150 mm Column 5 µm WAT106187 1860002934.6 x 150 mm Column 5 µm WAT106190 1860002944.6 x 250 mm Column 5 µm WAT106184 186000295

3.9 x 150 mm Cartridge* 5 µm WAT106181 —4.6 x 150 mm Cartridge* 5 µm WAT106175 —4.6 x 250 mm Cartridge* 5 µm WAT106178 —

* Require endfittings, see below

Symmetry300™ Column and Cartridge ColumnMethod Validation Kits Three Columns from Three Different Batches to Test Reproducibility.

13

Waters Cartridge Column

Waters cartridge system requires re-useable endfittings. These cartridge endfit-tings need to be ordered when purchasing a Waters HPLC cartridge columnfor the first time. These cartridge columns should be connected to your HPLCinstrument using Waters standard connectors.

Disposable Cartridge

Reusable End Connectors

Waters Cartridge Column

ConnectorEndfitting

WAT037525

C-Clip

GrooveRetaining

Nut

Cartridge

Waters Sentry™ Guard Column andUniversal Guard Holder

Waters offers a line of Sentry™ Guard columns that contain the same high performance packing materials as all of the Waters analytical columns.Developed for customers needing increased capacity for biological fluid clean-up and/or temperature control, the Sentry™ Guard column is available in twoholder designs--one to be used as an integrated part of the Waters steel cartridge column with reusable endfitting, the other for use with any HPLC column. Both are installed without tools.

Sentry™ Guard Column

Integrated Guard Holder

Universal Guard Holder

Cartridge End-Fittings

2.1 x 50 mm, 2.1 x 100 mm, 2.1 x 150 mm, 2.1 x 250 mm use Part Number 7000001173.0 x 50 mm, 3.0 x 100 mm, 3.0 x 150 mm, 3.0 x 250 mm use Part Number WAT0375253.9 x 50 mm, 3.9 x 100 mm, 3.9 x 150 mm, 3.9 x 250 mm use Part Number WAT0375254.6 x 50 mm, 4.6 x 100 mm, 4.6 x 150 mm, 4.6 x 250 mm use Part Number WAT037525

Sentry™ Guard Column

© 2002 Waters Corporation. Symmetry, Symmetry300, SymmetryShield, SymmetryPrep, Alliance and Watersare trademarks of Waters Corporation. IntegraFrit, PicoFrit, and PicoTip are trademarks of New Objectives.720000454EN

Sales Offices:

Austria and Export (Central and South East Europe, CIS, Middle East, India and India Subcontinent)(43) 1 8771807Australia (61) 2 9933 1777Belgium and Luxembourg (32) 2 7261000Brazil (55) 11 5543 7788Canada 800 252 4752China (86) 10 6526 0828Czech Republic (42) 02 617 11384Denmark (45) 46 598080Finland (358) 9 506 4140France (33) 1 30 48 72 00Germany (49) 6196 40 06 00Hong Kong (852) 2964 1800Hungary (36) 1 350 5086India (91) 80 8371900Ireland (44) 208 238 6100Italy (39) 02 27421.1Japan (81) 3 3471 7191Malaysia (60) 3 704 8600Mexico (5255) 5524 76 36The Netherlands 31 (0) 76 508 72 00Norway (47) 638 46 050Poland (48) 22 883 4400Puerto Rico (787) 747 8445Russia/CIS (7) 095 931 9193Singapore (65) 278 7997Spain (34) 93 600 93 00Sweden (46) 8 594 69 200Switzerland (41) 62 889 2030Taiwan (886) 2 543 1898United Kingdom (44) 208 238 6100All other countries: Waters Corporation U.S.A.508 478 2000/800 252 4752

Literature CodeSymmetry® Application Notebook WN033SymmetryShield™ Application Notebook WN035SymmetryShield™ White Paper 720000207EN

To request your free copy, or for more information,please contact your local Waters Sales Office.

The quality management system of Waters’ manufacturing facility, Taunton, Massachusetts, andWexford, Ireland complies with the International Standard ISO 9002 Quality Management andQuality Assurance Standards. Waters’ quality management system is periodically audited by theregistering body to ensure compliance.

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