Tiny Parts That Can Make or Break an HPLC Separation (Pun … · 2017. 3. 9. · 4.6 x 50 mm, 1.8...

ESR Pharmaceutical Applications, Clogs

CLOG00.GAL

Plumbing Connections and Fittings:Tiny Parts That Can Make or Break an HPLC Separation

(Pun Intended)

Dispersion Reduces HPLC Performance

What Causes Dispersion?

• Original Sample Concentration being diluted as it is carried

through the system plumbing (Extra Column Volume)

What Increases Dispersion?

• Connecting Tubing that is too long

• Connecting Tubing that is to large in diameter

• Connections that have gaps and form small mixing chambers

Instrument Impact on Column Performance

.

Extra-column

Volume

.

Number of Scans

or points

3

As Efficiency Increases Peak Width DecreasesSystem Dispersion Becomes More of a Factor

Columns: Eclipse Plus C18, as described below. Mobile Phase: A: water, B: MeOH, (15:85) Injection volume: 6uL

Temperature: 25°C Flow: 1 mL/min. Detection: 310, 4 nm, 0.5 s response time, semi-micro flow cell, Sample: Sunscreens

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

min0 2 4 6 8 10 12 14

mAU

0

20

40

60

80

100

min0 2 4 6 8 10 12 14

mAU

0

50

100

150

4.6 x 100 mm, 3.5 µm

1

2 3

4

Rs3,2= 6.65

Rs3,2= 6.51

Rs3,2= 6.41

4.6 x 50 mm, 1.8 µm

4.6 x 150 mm, 5 µm

Approximate Extra Column Volume Limits

for This 4.6mm i.d. Column Example

5um 3.5um 1.8um

Peak (1) Width in uL 181 136 45

Injection (1/10 P.V.) 18 14 5

Extra Column (1/3 P.V.) 60 45 15

ECV minus Inj. Vol. 42 21 10

ECV Estimate for

2.1mm i.d. Column

8 4 2

Tubing Volume

Tubing Length 10mm 50mm 100mm 150mm

Tubing i.d. Volume Volume Volume Volume

0.17mm (green) 0.227 uL 1.1uL 2.27 uL 3.3 uL

0.12mm (red) 0.113 uL 0.55uL 1.13 uL 1.65 uL

Group/Presentation Title

Agilent Restricted

Month ##, 200X

Dispersion in the Tubing

Dispersion Calculation

σt2 = π2r6Lu/24Dm

Dispersion in the tubing is proportional to the

• Length of tubing

• 6th power of the tube radius

Shortest tubing lengths possible minimize dispersion

Small changes in tubing i.d. have major effects on

peak width and efficiency

Conversion for Fast and Ultra-Fast HPLC1200 through 1260 Series LC Systems

High pressure

Gradient pump

Std or Well

Plate sampler

Diode Array

Detector

Standard assembly

without standard mixer

0.12 x 400 mm capillary

Mass

Spectrometer

0.12 x XX mm PEEK Capillary

High pressure

Gradient pump

Std or Well

Plate sampler

Rapid Resolution

HT Column

Diode Array

detector

Waste




3 L

heat exchanger

Thermostatted

Column

compartment

Traditional LC Columns Fast LC/UHPLC Columns

Rapid Resolution

HT Column



3 L

heat exchanger

Thermostatted

Column

compartment

8

Optimizing Connecting Tubing Volume

For UHPLC Columns

min0.5 1 1.5 2 2.5

mAU

0

100

200

350

400

550

600 System Tubing Volume Optimized

0.12mm i.d. tubing

Peak width 0.018 min


Resolution 1.902

min0.5 1 1.5 2 2.5

mAU

0

100

200

300

400

System Tubing Volume Not Optimized

0.17mm i.d. tubing



Resolution 0.961


CLOG02.GAL

Why Do We Disconnect and Reconnect

Plumbing Fittings in an HPLC or UHPLC?

• Line Is Plugged

• Replace a Column

• Rearrange Instrument Configuration


CLOG12.GAL

1 2

3

Clogging at the Column Head


CLOG13.GAL

Column Protection

Inline Filter

Inexpensive < $5

Somewhat Effective

Guard Column

More Expensive $10 - $100

More Effective

Analytical Column

Expensive $200 - $1000The

Column

Is the Most

Effective

Filter in the Whole LC

13

Stainless Steel and Polymer FittingsWhich type is used and when?

Stainless Steel (SS) fittings are the best choice for reliable high pressure sealing

• Agilent uses Swagelok type fittings with front and back ferrules – which give best sealing performance –throughout all our LC systems

PEEK (<400 bar System Pressure) fittings are ideal where:

• Connections are changed frequently, i.e. connecting columns

• Pressure is less critical

PolyKetone

• Easy, hand tighten column connection

• 600 bar Pressure Rating PN: 5042-8957 (10/pk)

• Fits to SS Tubing

ESR Pharmaceutical Applications, Leaks

LEAK00.GAL

Swagelok

Waters

Parker

RheodyneValco

Uptight

0.250 in.

0.520 in.

0.220 in.

0.300 in.

0.250 in.

0.210 in.

0.320 in.

0.225 in.

Troubleshooting LC Fittings, Part I. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988)

STEVE

GEORGE Compression Screws from

Various Manufacturers


LEAK02.GAL

STEVE

GEORGE

Nut

tooshort

Ferrule

not

compressed

1 2Fittings

matched

Extra

Threads are

OK

3

Mismatched Compression Screws

16

Column Connectors Used in HPLC

0.090

in.

0.130

in.

0.090

in.

0.170

in.

Swagelok Waters

Parker Rheodyne

Valco Uptight

0.090

in.0.080

in.

Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch. LC/GC Magazine 6:788 (1988)


LEAK03.GAL

STEVE

GEORGE Mismatched, Swaged Fittings

Too

Short

Just

Right

Too

Long

min0 0.1 0.2 0.3 0.4

mAU

0

20

40

60

80

100

120

140One bad capillary connection!

min0 0.1 0.2 0.3 0.4

mAU

0

30

60

90

120

150

180

210 Fixed!

130 mAU

160 mAU

Influence of Bad Post-Column Connection

Tubing After CuttingEven “properly cut” Tubing Has Problems

• Best Practice is to use “pre-cut” tubing (capillary in Agilent speak)

• Second Best – use a high quality tubing cutter

• DO NOT USE Side Cut Pliers or Scissors” (You Laugh, but it has happened)


LEAK06.GAL

Polymeric Fittings

Not a Recommended Tool for Fittings!

And Not This One!


LEAK05.GAL

Over Tightening Spells Disaster!

Properly Tightened PEEK Fitting/Tube

View of the “crimp” Needed to Hold the Tube

In a High Pressure Fitting

This Adjustable Wrench is

WAY TOO BIG

for a ¼” Nut

These Are Acceptable Tools for SS Fittings!

Make Sure The Pieces Are Assembled Properly

Proper Tightening

• Hold Column in one hand

• Hold Capillary/Nut/Ferrule Assembly in other hand

• Keep Capillary “bottomed” while hand tightening the nut

• Tighten with Wrenches ¼ turn (wrench on end fitting and nut only)

• DO NOT OVERTIGHTEN!!!!

Do NOT Use the Flats on the Column Barrel

Make sure parts are “locked” in


LEAK04.GAL

Note slight

of ferrule.

Excessive

Good

Fit

Bad

Fit

Overtightening

permanently damages

fittings and leads to

• Leaks

• Damage to new

columns

• Poor poeak shape

Zero Dead Volume Fitting

STEVE

GEORGE

joint with same I.D.

Capillaries abut on

Untightened

Surfaces

not

forced together

curvature

Tightening FittingsFinger tight, then 1/8 to 1/4 turn

Confidential

LE

SS

ST

RE

SS

MO

RE

SU

CC

ES

SA

gile

nt A

-Lin

e L

C s

up

plie

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March 8, 2017

33

The Agilent A-Line FittingsImportance of the Spring Loaded Feature

Most commonly used fittings in UHPLC are non-adjustable 2-piece or 3-piece metallic fittings. Since different manufacturers of column hardware have different design in column end fittings, as shown in Figure 1, a new set of tubing and fittings needs to be installed for every brand of column to guarantee that the stem length, namely the length between the bottom of the ferrule and the end of tubing, fits the column end fitting.

The spring-loaded design constantly pushes the tubing against the receiving port, delivering a reproducible connection with no dead volume for consistent chromatographic performance

Stem length is adjustable through the spring, which makes the fitting compatible with all types of LC columns

The Agilent A-Line Quick Connect Fitting3 Single Steps to Connect a column

March 8, 2017

34

LE

SS

ST

RE

SS

MO

RE

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CC

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C s

up

plie

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• We have successfully tested the fittings on UHPLC columns from Agilent,

Waters, Supelco and Phenomenex.

• The fittings can be used in non-Agilent instruments on all Swagelok

compatible connections.

Note: The fittings must be used with A-Line capillaries, which are not

obligatory compatible with capillaries used in non Agilent instruments.

Confidential

1

3

2

The Agilent A-Line Quick Connect FittingRobustness

March 8, 2017

35

LE

SS

ST

RE

SS

MO

RE

SU

CC

ES

SA

gile

nt A

-Lin

e L

C s

up

plie

s

Monitoring of tailing factor and theoretical

plates number over 200 reconnections of

A-Line Quick Connect Fitting.

Chromatogram overlap before and after 200

reconnections

• No visible change of chromatogram after 200 reconnections.• Tailing factors and theoretical plates stayed constant within the

experimental allowance through the reconnection procedures.

Fitting connection kept dead-volume free and leak-free after 200 times of reuses

Confidential

Let’s PLAY!

Tiny Parts That Can Make or Break an HPLC Separation (Pun … · 2017. 3. 9. · 4.6 x 50 mm, 1.8...

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