Slide 1Chairperson: John Vis

Optimizing Your Purification System for Highest Recovery

Presented by: Udo Huber

• 1995 PHD in organic chemistry from the University Karlsruhe/Germany• 1996 - 97 Postdoctoral fellow at the University of Hawai’i at Manoa• Since 1997 Application Chemist with HP/Agilent• Since 2000 Senior Application Chemist for the purification system and valve solutions

Slide 2Chairperson: John Vis

Contents

• Delay volume calibrationFraction delay sensor

• What is detector delay?Signal filtering

• What is system delay?The CAN network: Integrated intelligence

• Influence of delay volume on recoveryDelay volume control with optimized design/setup

Slide 3Chairperson: John Vis

Purification system schematics

Fraction container

Detector

Divertervalve

Waste

VD2 / tD2

Fraction collector

VD1 / tD1

Slide 4Chairperson: John Vis

Fraction delay – UV-based fraction collection system

min0 1 2 3 4 5 6 7 8 9

mAU

200

600

1000

1400

t0 tE

Fractioncontainer

Detector

Divertervalve

Waste

VD2 / tD2

Fraction collector

VD1 / tD1

Start of fraction collection ⇒ when start of peak arrives at diverter valveStart of fraction collection: t0 + tD1

End of fraction collection ⇒ when end of peak arrives at needle tipEnd of fraction collection: tE + tD1 + tD2

Slide 5Chairperson: John Vis

Fraction delay sensor (FDS)

The FDS is a detector in the fraction collector. In combination with UV and MS detection of a peak, it allows accurate calculation ofthe time delay between peak detection and fraction collection.

Slide 6Chairperson: John Vis

Fraction delay sensor

The delay calibrant contains two compounds. One is detectable by UV and by the FDS and the other is detectable by the MS. With no column in place the compounds do not separate. The time difference between their detection in the UV, FDS and MS allows accurate calculation of the delay volumes.

Light source: GaAs Red LED Lamp (λmax = 654 nm)Detection: Si Photo detector (λ = 580 - 700nm)Delay Calibrant: Fast Green FCF for UV detector and FDS(G1946-85020) Caffeine for MSD

Slide 7Chairperson: John Vis

Fraction delay – UV-based fraction collection system

Diverter valveDetector

min0 1 2 3 4 5 6 7 8 9

mAU

200

600

1000

1400

min0 1 2 3 4 5 6 7 8 9

FDS

0.2

0.6

1.0

1.4

tD UV

FDS

VD1/tD1Inject delay calibration sample (no column)

Waste

VD2/tD2VD3: Internal volume of

FDS, tubing etc. VD3/tD3

Fraction delay sensor (FDS)

VD1 = (v • tD) - VD2 - VD3• v: flow rate•

Slide 8Chairperson: John Vis

Fraction delay – MS-based fraction collection system

Divertervalve

min0 1 2 3 4 5 6 7 8 9

mAU

0200

600

1000

1400

min0 1 2 3 4 5 6 7 8 9

TIC

020000

60000

100000

140000

min0 1 2 3 4 5 6 7 8 9

FDS

00.2

0.6

1.0

1.4

tD

UV

MSD

FDS

Make-uppumpDetector

Inject delay calibration sample(no column)

Splitter MSD

Waste

Fraction delay sensor (FDS)

Slide 9Chairperson: John Vis

ChemStation software –delay calibration

Set up your purification method and save it (MBFC only!)

Go to ChemStation Diagnosis view

Start Delay Volume Calibration

Slide 10Chairperson: John Vis

ChemStation software –delay calibration

Use a pre-defined calibration method (or select you own method using the Change method button), put the Delay Sensor Calibrant(Part No. G1946-85020) in vial position 1 and press Continue

Slide 11Chairperson: John Vis

ChemStation software –delay calibration

Review the results and transfer them automatically using the Calibrate button

Slide 12Chairperson: John Vis

ChemStation software –delay calibration

Calibration done and result transferred to the fraction collector configuration

Slide 13Chairperson: John Vis

Break Number 1

Please type your

question into the

Question Box at any time

during the presentation.

Slide 14Chairperson: John Vis

What is a detector delay time?

min2.5 2.6 2.7 2.8

mAU

0

5

10

15

20

25

min2.5 2.6 2.7 2.8

mAU

0

5

10

15

20

25

Signal filtering

Signal measured in the detector Signal displayed in ChemStation

Slide 15Chairperson: John Vis

What is a detector delay time?

min2.5 2.6 2.7 2.8

mAU

0

5

10

15

20

25 1. Datapoints measured2. Datapoints averaged3. Datapoint drawn

min2.5 2.6 2.7 2.8

mAU

0

5

10

15

20

25

Delay between actual measurement point and point drawn in software⇒ Detector delay

Slide 16Chairperson: John Vis

Detector delay times for different filtering/peakwidth settings

Peakwidth (filtering) must be smaller then peakwidth of narrowest peak in chromatogram. Otherwise 2 peaks could be “filtered” together to give a single peak.

DAD/MWD VWDPeakwidth[min]

Responsetime [s]

Signaldelay [s]

Peakwidth[min]

Responsetime [s]

Signaldelay [s]

< 0.01 0.1 0.05 < 0.005 < 0.1 0.07> 0.01 0.2 0.15 > 0.005 0.12 0.14> 0.03 0.5 0.5 > 0.01 0.25 0.29> 0.05 1.0 1.25 > 0.025 0.5 0.58> 0.10 2.0 2.75 > 0.05 1 1.31> 0.20 4.0 5.9 > 0.1 2 2.84> 0.40 8.0 11.9 > 0.2 4 5.97> 0.85 16.0 23.9 > 0.4 8 12.3

Slide 17Chairperson: John Vis

Detector delay time

• Additional detector delay time for DAD, MWD and VWD is automatically adjusted by the detector firmware.

• tdetector < tD1

• Recommendation: Peakwidth should be set to the second-lowest setting for the UV detectors.

Slide 18Chairperson: John Vis

What is a system/computer delay?

1. Detector sends signal to PC (LAN)2. PC processes signal and acknowledges a peak (PC)3. PC gets flow rate from pump (PC)4. PC calculates delay time (PC)5. Sends signal to fraction collector (LAN)

Fraction Collector

Detector

Sampler

Pumps

Detector

LAN network

Additional system/computer delay due to:• LAN traffic• CPU usage

Slide 19Chairperson: John Vis

The CAN network: Integrated intelligence

1. Detector detects peak2. FC acknowledges DAD peak (CAN)

- Looks up DAD delay volume (CAN)- Gets current flow rate from pump (CAN)- Calculates DAD delay time

3. Delays for delay time4. STARTS FRACTION COLLECTION (CAN)

Fraction Collector

Detector

Sampler

Pumps

Computer only:• monitors• displays results

LAN network

Slide 20Chairperson: John Vis

Break Number 2

Please type your

question into the

Question Box at any time

during the presentation.

Slide 21Chairperson: John Vis

Dispersion for different delay volumes

min0 0.5 1 1.5

0

50

100

150

200

250DAD Signal

FDS Signal

+ 0 µl + 25 µl + 50 µl + 100 µl+ 150 µl+ 200 µl

Fractioncontainer

Detector

Divertervalve

Waste

VD2 / tD2

Fraction collector

VD1 / tD1

Delay volume added with 0.25 mm I.d. tubing

Slide 22Chairperson: John Vis

Peak loss due to dispersion

min0 0.5 1 1.5

0

50

100

150

200

250DAD Signal

FDS Signal

+ 0 µl+ 50 µl + 100 µl

+ 200 µl

Delay volume added with 0.25 mm i.d. tubing

Slide 23Chairperson: John Vis

Resolution and peak width

min0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0

50

100

150

200

250

300

350

DAD Signal

FDS SignalIncreasing capillary length

Slide 24Chairperson: John Vis

Resolution and peak width

0 50 100 150 200 250

Added delay vol. [µl]

0.25 mm I.d.

0.8 mm I.d.

Reso

lutio

n

Resolution at detector

Resolution at fraction collector

• Standard fraction collector AS with 0.25 mm i.d. capillary from DAD to AFC gives point at 0 delay volume added

• Delay volume was enhanced using a 0.25 mm i.d. capillary or a 0.8 mm i.d. capillary, respectively.

• Dispersion with 0.8 mm i.d. capillary higher for same delay volume due to squared influence of capillary radius on dispersion.

Slide 25Chairperson: John Vis

Does it really matter?

"If they [the chemists] want to purify a sample they'll know which one is best! The choice is a system that can actually separate and purify or one that sends e-mails with data the chemists can manipulate."

“[My customer] is achieving recoveries right in line with […]’s presentation [..] (not quite 110%). His quote: ”You guys don't know what you have here" was based …”

“Using a combined inject/collector seriously compromises the pluming of the system and leads to additional broadening and therefore loss of recovery. At […] we see 90% recovery with your system and 60% with others.”

Slide 26Chairperson: John Vis

Optimizing your purification system

• Correct delay volume calibrationFraction delay sensor vs. stop-watch method

• No timing error due to detector delayFirmware adjustment

• No timing error due to PC/LAN delayDirect LAN connection, intelligent CAN network

• Minimizing dispersion with optimized delay volumeDelay volume control with optimized design/setup