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Agilent A-Line UHPLC Fittings

Technical Overview

Introduction

Chromatographers working with HPLC and UHPLC are often challenged by problemssuch as peak tailing, peak broadening, split peaks, carryover, and so forth. Onecommon cause for those problems that is often overlooked and costs much time introubleshooting is poor tubing connection. Dead volume or micro-leakage in tubingconnections can greatly affect the performance and reproducibility of chromatographicanalysis, especially with modern UHPLC and Fast LC columns [1-2].

Fitting connection requirements

Fitting connections can have a very large impact on the peak shape of analytes.An ideal fitting connection should feature:

• Zero dead volume between tubing and receiving port

• Ability to remain free of leaks under ultra-high pressures and elevatedtemperatures

• Robustness over long-term use, preventing tubing slippage

• Ease-of-use

STOP THE PRESSES - AgilentQuick Connect fittings shortlistedfor the prestigious R&D 100sawards 2015. For more than50 years, the editors of R&Dmagazine have chosen the 100“most technologically significantproducts introduced into themarketplace over the past year”.Once again, an Agilent product isin line for this mark of excellence.

A-Line Quick Connect won TheAnalytical Scientist InnovationAward 2014 because it is the onlytrue finger-tight UHPLC fittingstable to 1,300 bar that can beused over and over again withoutloss of performance.

AWARDWINNERTASIA 2014

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Existing products

Nonadjustable metallic fittingsMost commonly used fittings in UHPLC are nonadjustable2-piece or 3-piece metallic fittings, which are permanent andnonadjustable after they have been assembled [3]. Sincedifferent manufacturers of column hardware use differentdesigns for column end fittings (Figure 1), a new set of tubingand fittings should be swaged for every brand of column. Thisensures that the stem length, namely the length between thebottom of the ferrule and the end of the tubing, is perfectlymatched (Figure 2C).

Figure 1. Common HPLC connectors from differentmanufacturers.

Figure 2. Comparison of correct and incorrect fittingconnections.

Swagelok

0.09 in

Upchurch

0.09 in

Rheodyne

0.17 in

Waters

0.13 in

Valco

0.08 in

Parker

0.09 in

A: Stem length too long → leak

B: Stem length too short → dead volume

C: Properly fitted tubing, no dead volume

min0.2 0.4 0.6 0.8 1.0

mAU

0

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40

60

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100

120

140

160 Zero-dead-volume connection with Agilent A-line Quick Connect fitting

Improperly installed stainless steel fitting with dead volume

Figure 3. The Agilent A-Line Quick Connect fitting deliverszero dead volume with improved peak shape.

If the stem length is too short, a dead volume is created,resulting in deterioration of peak shape, lower resolution, andcarryover (Figures 2B and 3). If the stem length is too long,the ferrule will not seat properly and leakage will occur(Figure 2A). In addition, conventional fittings and ferrules areoften over-tightened when wrenches are used, resulting inthe fitting getting permanently stuck in the column.

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Adjustable finger-tight fittingsTo solve the problems of conventional fittings, adjustablefinger-tight fittings have been developed that are compatiblewith different columns. These fittings usually have polymer(for example, PEEK) ferrules, which make the fittings reusablebecause the ferrule is not permanently attached to the tubing.However, many of them still have some drawbacks, such as:

• The inability to reach 1,300 bar ultra-high pressureswithout tools

• The need to follow strict guidelines on the exact torqueor range of turning angle to avoid over-tightening

• The need to check for leaks every time after reconnection

• The fitting often has to be retightened.

• If the polymeric ferrule does not grip the tubing stronglyenough, it could slip off the receiving port at ultra-highpressures or pressure cycling, leading to the creation ofdead volume.

Agilent A-Line fittings

Agilent A-Line UHPLC fittings avoid these drawbacks,enabling a reproducible and leak-free column connection.There are two types of A-Line fittings. The A-Line QuickConnect fitting (Figure 4A) is for column connections with1,300 bar sealability without the need for a wrench. TheA-Line Quick Turn fitting (Figure 4B) is for various flow pathconnections, including column inlet/outlet, valve, and otherconnections. This fitting seals up to 600 bar by fingertightening (depending on users and positions of connection)and to 1,300 bar with a wrench.

Figure 4. Agilent A-Line fittings. A) QuickConnect fitting, B) Quick Turn fitting.

A

B

The spring constantly pushes the capillary towards the receiving port.

Guaranteedzero dead volume

Figure 5. Unique spring-loaded design of Agilent A-Linefittings.

Hand-tighten the blue nut untilfeeling the first resistance.

Depress the lever, 1,300 bar tight.

Figure 6. Installation of Agilent A-Line Quick Connect fitting.

Both types of fittings have a novel spring-loaded design(Figure 5) that constantly pushes the tubing against thereceiving port, delivering a reproducible connection with nodead volume for consistent chromatographic performance.The stem length is adjustable through the spring, whichmakes both fittings compatible with all types of LC columns.In addition, the A-Line Quick Connect fitting has a uniquelever-actuated design, so that the spring assembly, includingthe lever, applies a constant force that presses the ferruleonto the tubing, avoiding tubing slippage. Little force isrequired to tighten the fitting to 1,300 bar (18,850 psi) withoutany tools. One merely tightens the nut by hand until feelingthe first resistance, then depresses the lever (Figure 6).

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Experimental

The performance of Agilent A-Line fittings was assessedusing an Agilent 1290 Infinity Binary LC. For robustness tests,the tested fitting (Agilent A-Line Quick Connect fitting ornon-Agilent UHPLC finger-tight fitting) was connected at thecolumn inlet. For compatibility testing A-line fittings withdifferent column brands, an A-Line Quick Connect fitting wasconnected at the column inlet, and A-line Quick Turn fittings(p/n 5067-5966) were used at the column outlet and detector.A mixture of uracil (10 µg/mL), phenol (200 µg/mL),4-chloronitrobenzene (25 µg/mL), naphthalene (40 µg/mL) inwater:acetonitrile 40:60 (v/v) was used as analyte.

Conditions to assess robustness and usabilityColumn: Agilent ZORBAX RRHD Eclipse Plus C18,

2.1 × 100 mm, 1.8 µm (p/n 959758-902)Mobile phase: A) Water

B) Acetonitrile; A:B 40:60 isocraticFlow rate: 1.4 mL/min (system pressure 1,100 bar)Injection volume: 1 µLColumn temperature: 40 °CDAD wavelength: 254 nm/4 nm, ref 400 nm/100 nm

Conditions to assess compatibility with different columnbrandsColumns: C18 phase, 2.1 × 50 mm, sub-2 µm columns from

different vendors Mobile phase: A) Water

B) Acetonitrile; A:B 45:55 isocraticFlow rate: 1.2 mL/min (system pressure 600 to 1,000 bar

depending on column)Injection volume: 1 µLColumn temperature: 25 °CDAD wavelength: 254 nm/4 nm, ref 400 nm/100 nm

Results and Discussion

Robustness over 200 reconnectionsTo assess the reusability and robustness of the Agilent A-LineQuick Connect fitting, the fitting was disconnected andreconnected 200 times. Chromatograms before and after200 reconnections are compared in Figure 7. It can be seenthat there was no visible change in the chromatogram after200 reconnections. The tailing factor of the peak with a longretention time (naphthalene) and theoretical plate numbers ofthe peaks with small retention factors (phenol and uracil)were monitored. They are the most sensitive measure forpeak-broadening effects through extra-column volume ormicro-leakage. Figure 8 demonstrates that the tailing factorsand theoretical plates stayed constant within experimentalvariables. This indicated that the fitting connection remainedfree of dead volume and leaks after 200 reconnections.

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0

20

40

60

80

100

120

First connection of Agilent A-Line Quick Connect fitting

After 200 reconnections140

mAU

Ura

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Phe

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4-C

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Nap

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Retention time (min)

Figure 7. Comparison of chromatograms before and after200 reconnections of an Agilent A-Line Quick Connect fitting.

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

0 50 100 150 200

Taili

ng fa

ctor

Number of reconnections

Tailing factor of naphthalene peak

Plates of phenol peak

Plates of uracil peak

Theo

reti

cal p

late

s

Figure 8. Monitoring tailing factor and the number oftheoretical plates over 200 reconnections of the Agilent A-Line Quick Connect fitting.

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Better robustness compared to fitting fromanother vendor The same experiment was repeated using a reusablefinger-tight UHPLC fitting with a polymeric ferrule fromanother vendor. After only 30 reconnections, the polymericferrule became locked onto the capillary and could not beadjusted, resulting in dead volume, as illustrated in Figure 2B.This was also confirmed by comparing the chromatograms inFigure 9, showing that deterioration of peak shape, includinglarger peak tailing and lower peak height, was evident after30 reconnections.

min0.1 0.2 0.3 0.4 0.5 0.6 0.7

mAU

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20

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60

80

100

120

Ura

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Phe

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4-C

hlor

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First connection of a non- Agilent UHPLC finger-tight fitting After 30 reconnections

Figure 9. Comparison of chromatograms before and after30 reconnections of a UHPLC finger-tight fitting from anothervendor.

Compatibility with different column brandsFitting connection design varies between different columnmanufacturers, and improper stem length of the fitting couldcause leaks or poor peak shape. To evaluate the compatibilityof Agilent A-Line fittings with different column brands,columns from three column manufacturers, Waters,Phenomenex, and Supelco, were connected usingAgilent A-Line fittings (Quick Connect fitting at the entranceand Quick Turn fittings at the exit and detector). The same setof A-line fittings and tubing was used for all columns. Ifcommon stainless steel fittings are used, a new set of fittingsand tubing is needed for each column brand to fit thegeometry of the column connection. The chromatograms withAgilent A-Line fittings were compared to those with properlyswaged stainless steel fittings in Figure 10. No visibledifference was observed, which indicates that the A-Linefittings work perfectly with all the tested column brands.

Figure 10. Compatibility of Agilent A-Line fittings withcolumns from different vendors.

min0.1 0.2 0.3 0.4 0.5 0.6

mAU

020406080

100120

min0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

mAU

020406080

100120140

Waters Acquity UPLC BEH C18 2.1 × 50 mm, 1.7 µm

2.1 × 50 mm, 1.7 µm

2.1 × 50 mm, 1.9 µm

Supelco Titan C18

Agilent A-Line fittingsIndividually swaged stainless steel fittings fitted to each column

min0.1 0.2 0.3 0.4 0.5 0.6 0.7

mAU

020406080

100120140

Phenomenex Kinetix C18

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Conclusions

Agilent A-Line fittings deliver usability, robustness, andUHPLC performance, with:

• Excellent chromatographic performance in terms oftailing factor, number of theoretical plates, and peakheight that remained constant through 200 reconnectionsof the fitting

• Zero-dead-volume connection guaranteed by the uniquespring-loaded design

• Compatibility with common column brands

• Ease-of-use

References

1. Rogatsky, E.;, Shaynah, B.; Cai, M.; Daniel, T. S.Optimizing UHPLC Fittings and Connections: A CaseStudy. J. Chromatogr. Sep. Techniq. 2013, 4, 193. doi:10.4172/2157-7064.1000193.

2. Fountain, K. J.; Neue, U. D.; Grumbach, E. S.; Diehl, D. M.Effects of extra-column band spreading, liquidchromatography system operating pressure, and columntemperature on the performance of sub-2-µm porousparticles. J. Chromatogr. A 2009, 1216, 5979-5988.

3. Majors, R. E. Fittings and Tubing for Ultrahigh-PressureLiquid Chromatography. LCGC North America 2014, 32,840-853.

Please watch the product video formore details

http://www.chem.agilent.com/en-US/products-services/Columns-Sample-Preparation/LC-LC-MS-Columns/Pages/greatconnections.aspx

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Ordering information for Agilent A-Line Quick Connect fittings

A-Line Quick Connect Fittings Part no.

Assemblies

Stainless Steel 0.075 × 105 mm 5067-5961












NOTE: Each assembly is equipped with a Quick Connect Fitting,a capillary, and a Swagelok fitting.

Replacement parts

Quick Connect LC Fitting 5067-5965

Front ferrule 5043-0924

Capillary Stainless Steel 0.075 × 105 mm 5500-1174
















NOTE: The A-Line Quick Connect fitting can only be equipped with anA-Line capillary specified in this table. The A-Line capillary is designedwith a spring and a holder.

www.agilent.com/chem

Agilent shall not be liable for errors contained herein or for incidental or consequentialdamages in connection with the furnishing, performance, or use of this material.

Information, descriptions, and specifications in this publication are subject to changewithout notice.

© Agilent Technologies, Inc., 2015Printed in the USASeptember 25, 20155991-5525EN

Ordering information for Agilent A-Line Quick Turn fittings

A-Line Quick Turn parts Part no.

Fittings and ferrules

Quick Turn UHPLC fitting 5067-5966

Front ferrule 5043-0924

Capillaries

Stainless steel, 0.075 × 105 mm long socket 5500-1198

Stainless Steel 0.075 × 150 mm long socket 5500-1232
















NOTE: A-Line Quick Turn fittings require the capillariesspecified in this table.

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