Tools for Speed and Selectivity - HPLC

Innovative LC Column Technology for

Formats, Selectivities and Tools for MaximizedTechnology for

Resolution and SpeedTools for Maximized Productivity

Paul BoguszewskiEMEAI Product Specialist

Novi Sad, Serbia

Outline

• Introduction

• Background to Zorbax particle technologyBackground to Zorbax particle technology

• Chemistry/Selectivity Overview

• Format overviewFormat overview

• Sub 2 micron technology (RRHD and RRHT)

– Speed vs Resolutionp

• Superficially Porous Technology (PoroShell)

– Invigorating older LC systems

41 Years ago….• Hewlett Packard acquires the assets of Hupe & Busch/Germany.• The HP1010 is the first ever HPLC Instrument available

• The HP1010 Liquid Chromatograph weighs more than half a ton.• The columns are packed with the latest 57 μm packing material.

Zorbax Base

Confused?

Too Many Columns!

ZORBAX Eclipse Plus Columns: First Choicef f•Eclipse Plus Excellent first choice for

method development

• Long life from pH 2-9g p

• Superior peak shape with basic compounds

• High resolution and efficiency with 1.8, 3.5 and 5 μm columns

• Rigorous QA/QC testing for greater long• Rigorous QA/QC testing for greater long-term reproducibility

Eliminate Tailing and Maximize Resolution with Eclipse Plus ColumnsEclipse Plus Columns

Eclipse Plus C18, 4.6 x 50mm1

Mobile Phase: 65% ACN: 35% 25 mM phosphate buffer (pH 7.4)

Tf = 1.20

1. Nortriptyline2 Di lth l t

Tf = 4.86 Competitive C18, 4.6 x 50mm

2. Dipropylthalate

1

min0 0.5 1 1.5 2

12

2

min0 0.5 1 1.5 2

Superior peak shape and better selectivity with Eclipse Plus means more resolution, easier quantitation and better results in your separations.q y p

ZORBAX StableBond Bonding: Low pH

•Superior low pH stability – down to pH 1 (C18)

•Non-endcapped for selectivity and lifetime

•Patented sterically protecting bonding

•6 different selectivities

•Ideal for most sample types at low pH

Stable Bond C18, 5um (pH 0.8, 90oC)

ZORBAX Extend-C18 : High pH

• High efficiency and long life at high pH – up to pH 11.5

• Unique bidentate bonding and double endcapping Unique bidentate bonding and double endcappingprovides high pH stability

• More efficiency and better peak shape than l b d lpolymer-based columns

•Excellent peak shape with double endcapping

•LC/MS at high pH (ammonium hydroxide) with high efficiency

Silica supportSilica support

Zorbax Extend C18: Carbonate Buffer pH 10.

ZORBAX Bonus-RP : Something Different!

• Excellent peak shape for challenging basic compounds at low and mid pH

• Unique reversed-phase selectivity

• Novel bonding technology with embedded polar group and steric protectionpolar group and steric protection

• Usable in 100% aqueous mobile phases

•Triple endcapped•Triple endcapped

Agilent Has Three Types of Columns for UHPLC

RRHT columns: 1.8um columns for fast LC up to 600 bar • Designed for compatibility with the Agilent RRLC • Compatible with some UHPLC’s with pressure limits up to 800 bar

RRHD columns: 1 8um columns for up to 1200 barRRHD columns: 1.8um columns for up to 1200 bar• Designed for compatibility with the new Agilent 1290 Infinity LC• Useable up to 1200 bar• Fully UHPLC compatible

Poroshell 120 columnsPoroshell 120 columns• 600 bar pressure limit for use with HPLC/UHPLC• UHPLC type performance but excellent fit for 400 bar LC’s

Sub 2um ColumnsSub 2um Columns

Van Deemter Equation

C term: Resistance to mass transfer is improved by Reducing particle size – this is the current standard approach Improving diffusion

hei

ght

H

ll ti l

The smaller the theoretical plate height H, the better the Resolution!

retic

al P

late

Resulting van-Deemter curveH = A + B/u + C x u

large particle small particle

Theo

r

C) Resistance to Mass Transfer

H min

B)Longitudinal DiffusionA) Eddy Diffusion

linear flow uu opt

Van Deemter Curve: HETP vs. Volumetric Flow Rate

0.0030Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50/30mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/min

H = A + B/u + Cu

0.0020

0.0025 Temp: 20°CSample: 1.0μL Octanophenone in Eluent

)

5um

0.0010

0.0015

HET

P (c

m

3.5um

0.0000

0.0005 1.8um

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Volumetric Flow Rate (mL/min)

Smaller particle sizes yield flatter curvesp yminimal shift to higher flow rates

Column Length & Particle Size

ColumnLength ( )

ColumnEfficiency N(5 )

ColumnEfficiency N(3 5 )

ColumnEfficiency N(1 8 )

Analysis Time*

(mm) N(5 µm) N(3.5 µm) N(1.8 µm)150 12,500 21,000 35,000

100 8 500 14 000 23 250Efficiency Analysis

-

33%100 8,500 14,000 23,250

75 6000 10,500 17,500

50 4 200 7 000 12 000

(N)

Pressure

Time

PeakVolume

-33%

-50%

67%50 4,200 7,000 12,000

30 N.A. 4,200 6,500

Volume -67%

-80%

90%

SolventUsage

15 N.A. 2,100 2,500 -90%

* Reduction in analysis time compared to 150 mm column; all columns 4.6-mm i.d.educt o a a ys s t e co pa ed to 50 co u ; a co u s 6 d

RR High ThroughputRR High Throughput

Rapid Resolution High Throughput

Parameter RRHT 1.8um, 600bar

RRHD1.8um, 1200 bar

Total Choices >140 >70

Bonded Phases 15 14

Lengths 150, 100, 75, 50, 30, 20mm 150, 100, 75, 50mm

Column ID’s 2.1, 3.0, 4.6mm 2.1 and 3.0mm, ,

Pore Sizes 80Å, 95Å, 80Å, 95Å, 300Å

Scalability to other particle sizes

Yes – 3.5, 5, 7um Yes – 3.5, 5, 7um

Recommended Choice for LC

Eclipse Plus C18, 4.6 x 50, 1.8um 959941-902


Recommended Choice for LC/MS

Eclipse Plus C182.1 x 50mm, 1.8um959741-902


Rapid Resolution HT Columns: Up to 20x Faster

Up to 20x faster than conventional HPLC • With same or better performance (resolution, precision, sensitivity, carry over)• Compliant with strictest regulatory performance requirements

HPLC, 40°CSB-C184.6 x 150mm, 5µm1.20ml/min, 40°CAnalysis Time = 11minAnalysis Time = 11min

min0 2 4 6 8 10

RRHT SB-C182.1mm x 50mm 1.8µm RRLC 40°C1.00ml/min, 40°CAnalysis Time= 1.1min

RRLC, 40 C10x faster

min0.2 0.4 0.6 0.8 10

RRHT SB-C182.1mm x 50mm 1.8µm2.40ml/min, 95°CAnalysis Time: 0.4min

RRLC, 95°C27x fasterPW = 197msec

0.2 0.60 0.4 min >20X faster

Reduce Analysis Times Dramatically

Long Term Stability at High Temp.

RR High DefinitionRR High Definition

What Can I do Beyond This with UHPLC?RRHD Columns for Use with the Infinity 1290 LC

Use a wider range of mobile phase choices

More viscous solvents – methanol, other alcoholsMobile phase additives to optimize separations (they increase viscosity)

Go faster – wider flow rate range!Go faster wider flow rate range!

Use longer columns – maximize resolution!

Optimize methods faster improve productivity!Optimize methods faster, improve productivity!

The 1290 Infinity Has a Wide Useable UHPLC Range

June 06, 2005

RRHD/RRHT Short 50mm Columns with Typical ACN Gradient Condition Don’t Require P > 600 barACN Gradient Condition Don t Require P 600 bar

Sample:Octyl dimethyl para-aminobenzoic acid, Column: RRHD Eclipse Plus C18 3 x 50mm 1.8um, Temp: 40C, Flow Rate: 1.5 ml/min, Gradient: 40-90% ACN/water over 2 minutes. Pressure: Up to 460 barPressure: Up to 460 bar

LC Beyond 600 bar Requires New RRHD ColumnsAgilent ZORBAX RRHT Eclipse Plus C18 2 1 x 100 mm 1 8 um; PN 959764‐902Agilent ZORBAX RRHT Eclipse Plus C18, 2.1 x 100 mm, 1.8 um; PN 959764‐902,

SN USUXU01664, LN B08101

1817 7 21 259

700

y = 935.21x + 7.1903

R2 = 0.9993

y = 1025.3x + 13.279

R2 = 0.9973

y = 1817.7x + 21.259

R2 = 0.9946

500

600

300

400

Pressure (b

ar)

Acetonitrile/Water(60:40)

200

300

Acetonitrile/Water(50:50)

Methanol/Water(60:40)

0

100

0 0.1 0.2 0.3 0.4 0.5 0.6

Flow Rate (mL/min)

Rapid Resolution High Definition

Parameter RRHD1.8um, 1200 bar

RRHT 1.8um, 600bar

Total Choices >70 >140

Bonded Phases 14 15

Lengths 150, 100, 75, 50mm 150, 100, 75, 50, 30,20mm

Column ID’s 2.1 and 3.0mm 2.1, 3.0, 4.6mm

Pore Sizes 80Å, 95Å, 300Å 80Å, 95Å,

Scalability to other Yes – 3.5, 5, 7um Yes – 3.5, 5, 7umyparticle sizesRecommended Choice for LC


Eclipse Plus C18, 4.6 x 50, 1.8um 959941-902302 902

Recommended Choice for LC/MS



Fast Separation with High Resolution on RRHD Column and Agilent 1290 Infinity LCColumn and Agilent 1290 Infinity LC

O t l di th l i b i idOctyl dimethyl para-aminobenzoic acid, RRHD Eclipse Plus C18, 2.1 x 100mm 1.8um, 40°C, 0 74 l/ i0.74 ml/min, 40-90% ACN/water over 4 minutes. Up to 775 bar

May 2009

Methanol Selectivity Can Be Explored When Operating Pressure is not a ProblemWhen Operating Pressure is not a Problem

Octyl dimethyl para-aminobenzoic acid, y y pRRHD Eclipse Plus C182.1 x 100mm 1.8um, 40C, 0.52 ml/min, 50-100% MeOH/water over 5.7 minutes. Up to 845 bar

Increase Flow for Fastest Separation on RRHD Use Complete Pressure Range of 1290 Infinity UHPLC

F = 2 ml/min T = 60°CF = 2 ml/minP = 975 bar

0.5min

mAU

300

400

500

T 60 CV_i = 0.5 µl x 100ppm eachWL = 275 nm DR = 160 Hz

min0 0 05 0 1 0 15 0 2 0 25 0 3 0 35 0 4 0 450

100

200

F = 2.3 ml/minP = 1110 bar 0.4min

ZORBAX RRHD SB-C18 2 1 x 50mm 1 8 µm

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45mAU

300

400

500

2.1 x 50mm 1.8 µm1200 bar

min0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

100

200

Maximizing Productivity by Maximizing Resolution

• Sometimes resolution is more important than speed!

• UHPLC offers many alternatives for increasing resolutionUHPLC offers many alternatives for increasing resolution.

• Speed can be maintained for some separations – will depend on sample complexity, flow rate etc.

Choose Column Length for Analysis Time and RsMobile Phase: 40:60 water:acetonitrile, Flow Rate: 0.5 mL/min, Temp: ambient, Columns: RRHD SB-C18, 2.1 mm ID, L th t d P k 1 il 2 t h 3 i h 4 b t h 5 l h 6 h h

RRHD 1.8um 1200 bar

Lengths as noted Peaks: 1: uracil, 2 acetophenone, 3 propiophenone, 4 butyrophenone, 5 valerophenone, 6 hexanophenone

Rs3,2: 5.64N6 : 10,700N6 : 10,700Pressure: 350 bar50 mm

min0 1 2 3 4

Rs3,2: 9.02N6 : 21,100Pressure: 566 bar

100 mm

Rs3,2: 11.98N6 : 34,100150 mm

min0 1 2 3 4

Pressure:750 bar

min0 1 2 3 40

The 150mm length RRHD column becomes a good option for high resolution with g g p gthe 1290 Infinity LC.

Gain Efficiency and Resolution with Long Length 150 + 100 mm = 250 mm 1.8um @ 1100 bar150 100 mm 250 mm 1.8um @ 1100 bar

α3,2: 1.196 42

150 mm (P:582 bar)

Mobile Phase 25:75 water:acetonitrile, Flow Rate: 0.5 mL/min, ambient temperature, ZORBAX RRHD SB-C18, 1.8um columns

mAU300

Rs3,2: 6.42N6 : 31,400Pw2: 0.016 minPw6: 0.029 min

50 ( 58 ba )

100

150

200

250

α3,2: 1.20Rs3 2: 8.76

150 +100 mm (P:1095 bar)1. Uracil2 Acetophenone

min0 0.5 1 1.5 2 2.5 3 3.5

0

50

mAU

250

300

Rs3,2: 8.76N6 : 50,200Pw2: 0.018 minPw6: 0.037 min

2. Acetophenone3. Propiophenone4. Butyrophenone5. Valerophenone6. Hexanophenone

50

100

150

200

250

High Rs in 3.5 minutes, 250mm, 1.8um at 1100 bar

min0 0.5 1 1.5 2 2.5 3 3.5

0

50

Resolution is fine on 150mm column, but for even more Rs longer column is used.

Where Does this Example Fall in the 1290 Infinity Range

This app falls in the “Power Range”pp g

Superficially Porous ColumnsSuperficially Porous Columns

Instrument Evolution

Legacy (<400bar)

• 3.5 and 5 micron

Modern UHPLC (600-1200 bar)

• Sub 2 micron

Method Transfer

Change in Column

SOP/ Dual Methods? €, $, £SOP/ Dual Methods? , $,

Poroshell 120 Columns for HPLC and UHPLC

• 80-90% efficiency of sub 2um

Poroshell 120 columns have:

• ~40-50% lower pressure

• 2.7um particle sizep

• 2um frit to reduce clogging

• 600 bar pressure limit• 600 bar pressure limit

The Poroshell particle has a solid core (1.7um) and a porous outer layer with a 0.5um diffusion path

Efficiency vs. 1.8μm

Comparable base loading to totally porous LC

What can be achieved?What can be achieved?

Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um

mAU100

11.5

96

Sulfadiazine,Sulfathiazole SulfapyridineSulfamerazine,Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine

Time %B0 833 3334 33Column: Eclipse Plus C18

Mobile Phase:A: 0.1% formic acid in WaterB: 0.1% formic acid in ACN

Time %B0 812 3313.2 33Column: 4.6 x 100mmPoroshell 120 EC C18

40

60

80

100

9.71

2

11.1

16

12.6

74

15.2

48

16.1

5116

.435

20.6

87

23.0

76

29.2

90

110 bar

Sulfamethoxypyridazine,SulfachloropyridazineSulfamethoxazole, Sulfadimethoxine

4.6 x 250mm, 5umFlow Rate: 1 mL/min

Poroshell 120 EC-C18, 2.7umFlow Rate: 1 mL/min

min5 10 15 20 25 300

20

mAU 7.03

7

32 b

200

250

1.71

99

2.31

12.

606

7 4.5

58

5.45

00

• Faster analysis (shorter column higher flow rate)• More Sensitive (smaller particle sharpens peak)• Better Optimized (faster analysis allows better tweaking)

325 bar

0

50

100

150 2.18

9

3.86

7

4.43

7

5.92

0

• Both run on Agilent 1100 G1315B DAD (under 400 bar)• No change in sample prep required, 2um frit used on both columns

min5 10 15 20 25 30

0

Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um

mAU100

11.5

96

See conditions previous slide

40

60

80

100

9.71

2

11.1

16

12.6

74

15.2

48

16.1

5116

.435

20.6

87

23.0

76

29.2

90

110 bar

min5 10 15 20 25 300

20

mAU

1 7.03

7

100

150

200

250

1.71

9

2.18

92.

31

2.60

6

3.86

7

4.43

7 4.55

8

5.45

0

5.92

0

325 bar

5

0

50

100 3

min

Best format for <400bar Instrument Range

USP Method Naproxen

USP Method Transfer: Simvastatin

PoroShell has 600bar Working Range!UHPLC S ti ith hi h i it l tUHPLC Separations with higher viscosity solvents

Growing PoroShell Range

Parameter RRHD1.8um, 1200 bar

RRHT 1.8um, 600bar

Poroshell 1202.7um 600,bar

Total Choices >70 >140 >80

Bonded Phases 14 15 8

Lengths 150, 100, 75, 50mm 150, 100, 75, 50, 150, 100, 75, 50, 30, 20mm 30mm

Column ID’s 2.1 and 3.0mm 2.1, 3.0, 4.6mm 2.1, 3.0, 4.6mm

Pore Sizes 80Å, 95Å, 300Å 80Å, 95Å, 120Å

Scalability to otherparticle sizes

Yes – 3.5, 5, 7um Yes – 3.5, 5, 7um Nearly identical selectivity to 3.5, 5um

RecommendedChoice for LC

Eclipse Plus C18, 3 0 100 1 8 m

Eclipse Plus C18, 4 6 50 1 8 m

Poroshell 120 EC-C184 6 100mm 2 7 mChoice for LC 3.0 x 100, 1.8um

959758-3024.6 x 50, 1.8um 959941-902

4.6 x 100mm, 2.7um695975-902

RecommendedChoice for LC/MS

Eclipse Plus C182.1 x 100mm, 1.8um

Eclipse Plus C182.1 x 50mm, 1.8um

Poroshell 120 EC-C183.0 x 100mm, 2.7um

959758-902 959741-902 695975-302

New Bonded Phases on Poroshell 120 O• Utilizes the same Eclipse Plus bonding process utilized in ZORBAX

Eclipse Plus Phenyl-Hexyl• Excellent choice for resolution of analytes such as aromatic

compounds, due to unique pi-pi interactions, or when other phenyl type columns are recommended

Poroshell 120 Phenyl Hexyl

• Embedded polar group provides unique selectivity for polar compounds• Often changes elution order of acidic and basic compounds• Stability from pH 2-9 allows for flexible method development across a

id H

Poroshell 120 Bonus-RP

wide pH range

• Proprietary bonded phase is an excellent choice for polar analytes that don’t retain well on C18StableBond chemistry enhances low pH stability

Poroshell 120 SB-AQ • StableBond chemistry enhances low-pH stability

• Good choice for polars where 100% aqueous mobile phase is needed. SB-AQ

• Bare silica HILIC (ships in HILIC solvents) for separations of polar dPoroshell 120 compounds

• Ideal for retention and analysis of polar compounds by LC/MS in high organic for maximum sensitivity

Poroshell 120 HILIC

These two bonded phases are for the analysis of very polar and polar basic compoundsThese two bonded phases are for the analysis of very polar and polar basic compounds.Use SB-AQ for LC-UV and HILIC for LC/MS

Comparison of Bonded Phases - 8 Steroids

mAU

50

100

150 Poroshell 120 EC-C18 13

2,5 6

4 78

0 2 4 6 8 10 12

0

mAU

50

100

150 Poroshell 120 SB-C18 13

2,5,64 7 8

0 2 4 6 8 10 12

0

mAU

50

100

150 Poroshell 120 Phenyl Hexyl1

3 7 842 5 6

0 2 4 6 8 10 12

0

mAU

50

100

150 Poroshell 120 Bonus RP 13,2 7,86,4

5

0 2 4 6 8 10 12

0

50

1.Hydrocortisone, 2.B Estradiole, 3. Andostadiene 3,17 dione, 4. Testosterone, 5. Ethyestradione 6 Estrone 7 Norethindone acetate 8 ProgestreoneEthyestradione, 6. Estrone, 7. Norethindone acetate, 8. Progestreone

Comparison of Bonded Phases - Beta Blockers

2mAU

100

200

300

1 34

5 6 7

Poroshell 120 EC-C18

min0 2 4 6 8 10 12-100

0

mAU

100

200

300

12

3 4 5 6 7Poroshell 120 SB-C18

min0 2 4 6 8 10 12-100

0

100

mAU

200

3002

3 45


min0 2 4 6 8 10 12-100

0

100

200 1 3 4 7 6

mAU

3002

5 Poroshell 120 Bonus RP

min0 2 4 6 8 10 12-100

0

100

200 1 3 45

7 6Poroshell 120 Bonus RP

1 Atenolol 2 Pindolol 3 Naldolol 4 Metoprolol 5 Acebutolol 6 Propranolol 7 Alprenolol1.Atenolol, 2. Pindolol, 3. Naldolol, 4. Metoprolol, 5. Acebutolol, 6. Propranolol, 7. Alprenolol

Separation of Planar Azo-DyesAU

DAD1 A, Sig=260,80 Ref=off (C:\DOCUMEN...INGS\WILLLONG\MY DOCUMENTS\DEC14\DATA\AZO DYES\SURVEY30000002.D)

min0 1 2 3 4 5 6 7 8 9

mAU

0100200300400

DAD1 A Si 260 80 R f ff (C \DOCUMEN INGS\WILLLONG\MY DOCUMENTS\DEC14\DATA\AZO DYES\SURVEY30000004 D)

Poroshell 120 EC‐C18

mAU

0100200300400


Poroshell 120 SB‐C18

min0 1 2 3 4 5 6 7 8 9

mAU

100200300400



min0 1 2 3 4 5 6 7 8 90

mAU

200300400


Poroshell 120 Bonus RP

15 to 100 %MeOH over 10 min @ 0 4 ml/min SolventA is 10 mM AmmoniumAcetate pH 4 8

min0 1 2 3 4 5 6 7 8 90

100200

15 to 100 % MeOH over 10 min @ 0.4 ml/min, Solvent A is 10 mM Ammonium Acetate pH 4.8

PoroShell Summary

• Extraordinary lot-to-lot reproducibility – Poroshell 120 columns are manufactured using a proprietary single-step porous shell process that dramatically reduces

minute differences between columns and lots

• Comparable speed and resolution to sub-2 μm columns- with up to 50% less backpressure – take HPLC and UHPLC performance to a new level of fl exibility and efficiency

• Superior peak shape – especially at pH 6-7 – for faster, more accurate results

• Long column life – Poroshell 120 columns use a standard 2 μm frit, and resist plugging with dirty samples

• Multiple bonded phases – C18s, C8s, Phenyl-Hexyl, and more! A growing family of bonded phases Multiple bonded phases C18s, C8s, Phenyl Hexyl, and more! A growing family of bonded phases for method development flexibility.

• Easy method transfer and scalability to ZORBAX bonded phases, for highest

productivity from lab to lab around the worldproductivity from lab to lab, around the world

• UHPLC guard column options that reduce your operating expenses by extending

the life of Poroshell 120 columns

Three Key Resources for Small Molecules Analysis

The LC Handbook5990-7595EN

LC Column Selection Guide5990-4435ENTo be updated 2012

LC Columns Poster5990-5325EN

5990 7595EN To be updated 2012

And the Newest On-Line Tool “The Navigator”: A Column and Sample Prep Selection ToolColumn and Sample Prep Selection Tool

“The Navigator” Can Provide Recommendations Based on the Users Best Starting Point

Get help and tech support will go to a special page to help customers get

Based on the Users Best Starting Point

immediate help in their language. They’ll also be prompted to provide feedback about the tooltool.

The Navigator provides recommendations based on information the chromatographer has at hand.

http://navigator chem agilent com OR: www agilent com/chem/navigatorhttp://navigator.chem.agilent.com OR: www.agilent.com/chem/navigator

Three Key Resources for Bio Molecules AnalysisHighlight Complete Range of Columns andHighlight Complete Range of Columns and

Recombinant Protein Characterization Guide5990-8566EN

Bio Columns Poster5990-5526EN Bio Columns Guide

5990-9384EN5990-8566EN

Method Translation Tool

Thank You for your Attention!

Questions?

Tools for Speed and Selectivity - HPLC

Documents

high ph high efficiency

types of columns

barrrhd columns

uhplcrrht columns

high ph ammonium hydroxide

carbonate buffer ph

phosphate buffer ph

columns rigorous qaqc