id i h G b C d C h d T f i d C ® C lB id i h G b UHPLC d HPLC E M h d T f U i F d C ® C lB id i th G b t UHPLC d HPLC E M th d T f U i F d C ® C lBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused Core® ColumnsBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused Core® ColumnsBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused-Core® ColumnsBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused-Core® ColumnsBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused Core® ColumnsBridging the Gap between UHPLC and HPLC: Easy Method Transfer Using Fused Core® Columnsdg g e G p be wee U C d C: sy e od s e Us g used Co e® Co u sg g p y gg g p y gg g g1 2S A S h 1 d T J W h 2S A S h t r1 nd T J W h 2S A Schuster1 and T J Waeghe2S A Schuster and T J WaegheS. A. Schuster and T.J. WaegheS. A. Schuster and T.J. WaegheJ gg

1Ad d M i l T h l I 3521 Sil id R d S i 1 K Q ill B ildi Wil i DE 19810 2MAC MOD A l i l I 103 C C Ch dd F d PA 193171Ad d M t i l T h l I 3521 Sil id R d S it 1 K Q ill B ildi Wil i t DE 19810 2MAC MOD A l ti l I 103 C C t Ch dd F d PA 193171Advanced Materials Technology Inc 3521 Silverside Road Suite 1 K Quillen Building Wilmington DE 19810; 2MAC MOD Analytical Inc 103 Commons Court Chadds Ford PA 19317Advanced Materials Technology Inc., 3521 Silverside Road, Suite 1-K, Quillen Building, Wilmington, DE 19810; MAC-MOD Analytical, Inc., 103 Commons Court, Chadds Ford, PA 19317Advanced Materials Technology Inc., 3521 Silverside Road, Suite 1 K, Quillen Building, Wilmington, DE 19810; MAC MOD Analytical, Inc., 103 Commons Court, Chadds Ford, PA 19317gy , , , Q g, g , ; y , , , ,g g g

C id i f M h d T f Gr di nt M th d C p ri n Chr t rConsiderations for Method Transfer I C fi i d G di M h d T f l Gradient Method Comparison Chromatograms R d ti f M th d T f fConsiderations for Method Transfer Instruments Configurations and Gradient Method Transfer Example Gradient Method Comparison Chromatograms Recommendations for Method Transfer fromAb

Co s de at o s o et od a s eMobile Mobile Instruments, Configurations and Gradient Method Transfer Example p g Recommendations for Method Transfer from

Abstract Mobile Phase A

Mobile Phase B

st u e ts, Co gu at o s a d Gradient Method Transfer ExampleA il t 1200 Shi d P iAbstract Phase A Phase B

pAgilent 1200 vs Shimadzu Prominence LC LC i h ALO C l

AbstractM th d P r t r S ttin T n f th d t A il nt 1100 nd Shi dz P in n S t

Agilent 1200 vs. Shimadzu Prominence UHPLC to HPLC with HALO Columns:Method Parameter Settings Transfer method to Agilent 1100 and Shimadzu Prominence Systemsg UHPLC to HPLC with HALO Columns:

C lMethod Parameter Settings Transfer method to Agilent 1100 and Shimadzu Prominence Systems

22

UHPLC to HPLC with HALO Columns:Many chromatographers are now exploiting the speed and efficiency advantages of UHPLC Column

g

1.42Many chromatographers are now exploiting the speed and efficiency advantages of UHPLC Degasser Pump A Autosampler Column Detector 1

0 42 mL/miny g p p g p y gp rt f th ir m th d d l pm nt ch m Th f l di p r i n UHPLC

Degasser Pump A Autosampler Heater Detector2 1 x 50 mm 0.42 mL/min

as part of their method development scheme. The use of low dispersion UHPLC Heater Flow rates and injection volumes are scaled as withOriginal Agilent 1200 Inst Method2.1 x 50 mm

290000 2 1 x 50 mmp p pi t t ti d hi hl ffi i t l ll th t diff t l mixer Heat Exchanger Flow Cell

Flow rates and injection volumes are scaled as with Original Agilent 1200 Inst. Method 761

1 µL No Delay290000 2.1 x 50 mm

UHPLC HPLCUHPLC HPLCinstrumentation and highly efficient columns allows them to screen different column mixer Heat Exchanger Flow CellShi dz i ti th d

Original Agilent 1200 Inst. Method

1.71 µL, No Delay UHPLC HPLCUHPLC HPLCinstrumentation and highly efficient columns allows them to screen different column

i h d l i di i ( H i difi ) h A il 1100 A il 1 00Shimadzu isocratic methods. 19 240000

UHPLC HPLCUHPLC HPLCstationary phases and analysis conditions (pH, organic modifier, temperature, etc.) much P B Parameter Agilent 1100 Agilent 1200

Shimadzu 2

0U 79 97 2.21

91stationary phases and analysis conditions (pH, organic modifier, temperature, etc.) much i kl h b f M i h hi h h l i hi h d d/

Pump B W tParameter Agilent 1100 Agilent 1200

P i C l 2 1 50 HALO C182 ID 10

0m

A

7 0.97

1.69 2

2.29

190000more quickly than ever before Moreover with this approach the resulting high speed and/orPump B Waste

g gProminence Column: 2.1 x 50 mm, HALO C18

ID m

.77 0

1

2 190000

itymore quickly than ever before. Moreover, with this approach the resulting high speed and/or Prominence Column: 2.1 x 50 mm, HALO C18

2

colIDFF

0

.941 nsi

Max Pressure: 600-1200 bar Max Pressure: 400 barMax Pressure: 600-1200 bar Max Pressure: 400 barq pp g g phigh resolution method will be more robust and efficient and will be able to generate 2

colFF 1. 140000

nte Max. Pressure: 600 1200 bar Max. Pressure: 400 barMax. Pressure: 600 1200 bar Max. Pressure: 400 bar

high resolution method will be more robust and efficient, and will be able to generate Press re Limit 12

colcol IDFF

8 036

12

In

l V l 15 L l V l 10 20 L i i d 35 55 L “ i ”l V l 15 L l V l 10 20 L i i d 35 55 L “ i ”g , g

analytical results that enable faster and better decisions with high productivity Pressure Limit 12

colcol ID .458 2.0

2.41

90000 Extracolumn Volume: ~7–15 µL Extracolumn Volume: ~10–20 µL optimized, 35–55 µL “as-is”Extracolumn Volume: ~7–15 µL Extracolumn Volume: ~10–20 µL optimized, 35–55 µL “as-is”analytical results that enable faster and better decisions with high productivity. Impact on Impact on 400 600 600 Mobile Phase A: water with 0 1% HCOOH 1

colID 0. 2 90000 µ

B d S di 5 10 L2µ p , µ

B d S di 5 10 L2 i i d 40 100 L2 “ i ”µ

B d S di 5 10 L2µ p , µ

B d S di 5 10 L2 i i d 40 100 L2 “ i ”y g p y Impact on Impact on

(b )400 600 600 Mobile Phase A: water with 0.1% HCOOH 1 col

7

Band Spreading: ~5–10 µL2 Band Spreading: ~5–10 µL2 optimized, 40–100 µL2 “as-is”Band Spreading: ~5–10 µL2 Band Spreading: ~5–10 µL2 optimized, 40–100 µL2 “as-is”

P I Gp

M h d T f P I Gp

M h d T f (bar) M bil Ph B ACN i h 0 1% HCOOH

257

301

374

833 40000

p g µ p g µ p , µp g µ p g µ p , µ

Parameter I G Method Transfer Parameter I G Method Transfer (bar) Mobile Phase B: ACN with 0 1% HCOOH 2

0

0.2

0.3

0.3

2.8

H h i i f h h d li l d iParameter I G Method Transfer Parameter I G Method Transfer Mobile Phase B: ACN with 0.1% HCOOH 2

LID

0

10000However, when it is necessary to transfer such methods to a quality control or production Column Heater G di t 3% ACN t 70% ACN i LID

0.0 1.0 2.0 ‐10000However, when it is necessary to transfer such methods to a quality control or production

i h d d l f i h h li i d il bili f UHPLC Pumping System • High pressure mixing systems typically C l mn H t r • Actual measured temperatures will Column Heater Bl k Bl k F d Ai Gradient: 3% ACN to 70% ACN in 22 colcol LIDVV

Time (min) 0.0 0.5 1.0 1.5 2.0 2.5 3.0

environment method developers often must cope with the limited availability of UHPLC Pumping System High pressure mixing systems typically Column Heater pi t t Block Block Forced Air Gradient: 3% ACN to 70% ACN in

2 7 i 22 colcolVV ( )

( ) HALO HALOHALO HALOenvironment, method developers often must cope with the limited availability of UHPLC • High Pressure have lower delay volumes, with less

F d i Y Yvary among instruments. Type

Block Block Forced Air 2.7min. 12 linjlinj VV Time (min.) HALO HALOHALO HALO

instrumentation and operator expertise in such laboratories Often it is necessary to transfer High Pressure N Y

y ,“ ndin ” f di nt p fil

• Forced air Y YM d t t t t h

Type 2.7min. 12 colinjcolinj LID

390000 3 0 x 50 mm • Minimize ECV by using minimal length of• Minimize ECV by using minimal length ofinstrumentation and operator expertise in such laboratories. Often, it is necessary to transfer Mixing N Y “rounding” of gradient profilesBl k h ( )

• Measured temperature may not match yp

Flo Rate: 0 42 mL/min 1112

llcolcol LID

3.0 x 50 mm • Minimize ECV by using minimal length of 2.1 mm L 2.1 mm L • Minimize ECV by using minimal length of 2.1 mm L 2.1 mm Lp p y

the method to a longer and larger ID column with a much larger particle size to be able tog

S t h d d t• Block heater (contact)

p ymethod set point Flow Rate: 0.42 mL/min. 11 colcol LID 340000 0 005” ID tubing in sample flow path0 005” ID tubing in sample flow paththe method to a longer and larger ID column with a much larger particle size to be able to • Low Pressure • Some systems have pressure-dependent

( ) method set point. Detector Type VWD DAD VWD/ 0.005 ID tubing in sample flow path.0.005 ID tubing in sample flow path.g g g p

transfer the method to conventional instrumentation (300 400 bar pressure limit)ow essu e

Mi i

p pdelay volume Detector Type VWD DAD VWD Max Pressure: 116 Bar 290000transfer the method to conventional instrumentation (300–400 bar pressure limit). Mixing delay volume. • Each detector brand and model can

yp Max. Pressure: 116 Bar • Use Flow Cell with volume of 1–5 µL• Use Flow Cell with volume of 1–5 µL( p ) g

D t t TEach detector brand and model can

i f i hi d l d T 45°C240000ty

Use Flow Cell with volume of 1 5 µLUse Flow Cell with volume of 1 5 µL

• Affects delay volume Detector Type vary in performance within model and Flow Cell Temperature: 45°C D l l f h i t t h ld b nsi

S D Ti C R Ti 1S D Ti C R Ti 1• Affects delay volume ypV D Y Y

y pwithin brand

Flow Cell 5 2 2 5

Temperature: 45 C Delay volumes of each instrument should be 190000ten

Without use of injection delay on HALO HALO • Set Detector Time Constant or Response Time1HALO HALO • Set Detector Time Constant or Response Time1

F d C ® UHPLC l i h 2 7 i l i d li f bl • Affects gradient shape and especially • VWD Y Y within brand.V l ( L)

5 2 2.5 D t ti DAD 275 B d idth 8y

d d d d

InWithout use of injection delay on HALO HALO pto fastest setting

HALO HALO pto fastest settingFused-Core® UHPLC columns with 2.7 µm particle size can deliver performance comparable Mixer Volume N Y • Affects gradient shape and especially

• Perform nce mon br nd c n r Volume (µL) Detection: DAD 275 nm, Bandwidth, 8 measured and if available injector delay should be 140000j y

Shi d h ito fastest settingto fastest settingFused Core® UHPLC columns with 2.7 µm particle size can deliver performance comparable

b 2 l 40 50% f h b k Thi b fi k h d fMixer Volume N Y

baseline noise with some solvents and • Diode Array (DAD) • Performance among brands can vary Volume (µL) Detection: DAD 275 nm, Bandwidth, 8 measured, and, if available, injector delay should be 90000Shimadzu system changes in 2 1 mm L 3 0 mm L2 1 mm L 3 0 mm Lto sub-2-µm columns at 40–50% of the back pressure This benefit makes method transfer

baseline noise with some solvents and ddi i ( TFA)

y ( )for noise sensitivity linearity Injection Volume: 2 0 µL used to correct the effective delay volume to that of 90000Shimadzu system changes in 2.1 mm L 3.0 mm L

• Set Data Rate 10 Hz2.1 mm L 3.0 mm L

• Set Data Rate 10 Hzto sub-2-µm columns at 40 50% of the back pressure. This benefit makes method transfer additives (e.g., TFA) for noise, sensitivity, linearityFl C ll

Injection Volume: 2.0 µL used to correct the effective delay volume to that of l ti

• Set Data Rate 10 Hz• Set Data Rate 10 Hz

much easier between UHPLC and HPLC systems It is only necessary to make( g , ) Flow Cell

j µS l S l 50 50 M OH/

yh i i l h d

40000resolution occur.much easier between UHPLC and HPLC systems. It is only necessary to make • Affects Extracolumn Dispersion • Flow cell design can affect dispersion

ow CeSample Solvent: 50:50 MeOH/water the original method.

10000

resolution occur.• For gradient separations, adjust column• For gradient separations, adjust columny y y

modifications to the extracolumn volume and dispersion of the HPLC system with some Tubing Volume Y Y• Affects Extracolumn Dispersion

Fl C llFlow cell design can affect dispersion i ifi l Path length 10 6 5

Sample Solvent: 50:50 MeOH/water the original method.‐10000 For gradient separations, adjust column

ilib i i id i l lFor gradient separations, adjust column

ilib i i id i l lmodifications to the extracolumn volume and dispersion of the HPLC system, with some Tubing Volume Y Y(aka Peak Variance) and delay volume Flow Cell significantly Path length 10 6 5

D R 40 H 0.0 0.5 1.0 1.5 2.0 2.5 3.0 equilibration time considering larger column equilibration time considering larger column p y ,additional adjustments to the analysis conditions

(aka Peak Variance) and delay volumeD i

g y

Fl ll hl h ff i d

g( )

Data Rate: 40 Hz. 0.0 0.5 .0 .5 .0 .5 3.0

HALO HALOq g g

l l HPLC d l l d hi hHALO HALOq g g

l l HPLC d l l d hi hadditional adjustments to the analysis conditions. Diff h l i i

• DesignY Y

• Flow cell pathlength affects noise and (mm)Data Rate: 40 Hz.

Time (min.) HALO HALO volume, larger HPLC delay volume, and higher HALO HALO volume, larger HPLC delay volume, and higher j yD l V l

• Differences can change selectivityg

V lY Y

p gsignal

(mm)Response Time: 0 02 sec VV 390000

, g y , gflow rate with larger ID column

, g y , gflow rate with larger ID columnDelay Volume N Y

g y

Aff i i i di• Volume signal. Response Time: 0.02 sec. VV UHPLCDHPLCD )()( 390000

3 0 L 3 0 Lflow rate with larger ID column.

3 0 L 3 0 Lflow rate with larger ID column.Delay Volume • Affects retention times in gradients

T • Mismatch of column and detector Mi Rp VV

T UHPLCDHPLCD )()( 340000 4 6 503.0 mm L 3.0 mm L3.0 mm L 3.0 mm L

W ill di h h b id d d dj d h f ig

• Temperature • Mismatch of column and detector Min. Response0 06 0 0 0 0 Flow Cell: 2 µL micro flow cell T UHPLCDHPLCD )()( 340000 4.6 x 50 mm • If necessary for gradient methods program an• If necessary for gradient methods program anWe will discuss the parameters that must be considered and adjusted when transferring • C n ff t tr l n di p r i n p

ptemperatures increases noise

Min. Response 0 062 0 02 0 02 Flow Cell: 2 µL micro flow cell Tinjdelay 290000

If necessary, for gradient methods, program an If necessary, for gradient methods, program an We will discuss the parameters that must be considered and adjusted when transferring i i d di h d f LC i i l LC Flow Rate Y Y

• Can affect extracolumn dispersion, esp. temperatures increases noise.Time (sec )

0.062 0.02 0.02P 600 b Finjdelay 290000 injection delay to reduce “effective delay volume”injection delay to reduce “effective delay volume”

isocratic and gradient methods from UHPLC instruments to conventional HPLC systems Flow Rate Y Yfor different instruments Time (sec.) Pressure max.: 600 bar Fj y

240000y

injection delay to reduce effective delay volumef h HPLC h h d l l f

injection delay to reduce effective delay volumef h HPLC h h d l l fisocratic and gradient methods from UHPLC instruments to conventional HPLC systems. for different instruments

Detector Settings • Inadequate data rate causes poor peak ( ) Pressure max.: 600 bar F 240000

sity of the HPLC system to match the delay volume of of the HPLC system to match the delay volume of g

Isocratic and gradient examples will be shown in which Fused Core UHPLC separations are Detector Settings q p pfid lit d b d ffi i 190000en

s y yh UHPLC

y yh UHPLCIsocratic and gradient examples will be shown in which Fused-Core UHPLC separations are

Injector Type • Can affect extracolumn dispersion, esp. • Data rate (pts/sec)fidelity, decreases observed efficiency, Maximum 80 Hz 190000

nte the UHPLC system.the UHPLC system.g p p

transferred to different HPLC instruments having different extracolumn dispersion and Injector Type Can affect extracolumn dispersion, esp. f diff t i t t

• Data rate (pts/sec)and increases tailing Maximum

13 7 H80 Hz

50 H • Standard mixer was removed 140000

In HALO HALOy

HALO HALOy

transferred to different HPLC instruments having different extracolumn dispersion and A t l

for different instruments • Response Timeand increases tailing. 13.7 Hz 50 Hz Standard mixer was removed

An injector program can be set on some instruments140000 HALO HALOHALO HALOg p

different dela ol mes G idance ill be offered to make s ch method transfers more• Autosampler

Fi d l h l di i h• Response Time Y Y • Bandwidth mismatch or use/absence Data Rate

13.7 Hz(40 Hz used)

50 Hz An injector program can be set on some instruments 90000 3 0 L3 0 Ldifferent delay volumes. Guidance will be offered to make such method transfers more

p(Fixed loop flow Y Y • Fixed loop has lower dispersion than • Detection wavelength

Y Y • Bandwidth mismatch or use/absence Data Rate (40 Hz used)All t bi i i l l th d 0 005” ID

j p gth t th di t t t d th l i i j t d

90000 3.0 mm L 4 6 mm L3.0 mm L 4 6 mm Lyf l

(Fixed loop, flow Y Y p pflow through needle type

• Detection wavelength of reference wavelength for DAD can( ) • All tubing was minimal length and 0.005” ID so that the gradient starts, and the sample is injected 40000

4.6 mm L4.6 mm Lsuccessful. through needle) flow through needle type

bandwidthof reference wavelength for DAD can

k i h d RIg g g , p j

T40000successful. through needle)

• Manual injectors often least dispersionbandwidth cause peak area ratio changes and RI after a defined time delay Ti jd l 10000• Manual injector • Manual injectors often least dispersion • Reference wavelength

p ganomalies respectively • Automatic delay volume reduction was turned ON

after a defined time delay, Tinjdelay ‐10000Manual injector Reference wavelength anomalies, respectively. • Automatic delay volume reduction was turned ON. j y0.0 0.5 1.0 1.5 2.0 2.5 3.0 Note: Response Time = 2.2 Time ConstantNote: Response Time = 2.2 Time Constant

Time (min )Note: Response Time 2.2 Time ConstantNote: Response Time 2.2 Time Constant

Time (min.)

T Ad t f I j ti D lG id f M i Fl R f HPLC Isocratic Method Transfer from Shimadzu to 1100 and 1200 Advantages of Injection Delay 4 611 NO inj delay R l f G di M h d T f fGuidance for Maximum Flow Rates for UHPLC Isocratic Method Transfer from Shimadzu to 1100 and 1200 Advantages of Injection Delay 0 04

4 2.6 NO inj. delay Results for Gradient Method Transfer fromGuidance for Maximum Flow Rates for UHPLC Isocratic Method Transfer from Shimadzu to 1100 and 1200 g j y

300

2.0 Results for Gradient Method Transfer from

Obj tiGuidance for Maximum Flow Rates for UHPLC 3 2

80 Results for Gradient Method Transfer from S d C l iObjectives 3.

042 Summary and ConclusionsObjectives

M h d b T f d HPLC i h HALO 9

3.1

4

Agilent 1200SL Binary A il 1200 A il 1100 d Shi d Summary and ConclusionsObjectivesMethods to be Transferred to HPLC with HALO Agilent 1100 QuaternaryShimadzu Prominence A il t 1200SL Bi 47

9 3.Agilent 1200SL Binary Agilent 1200 to Agilent 1100 and Shimadzu Summary and ConclusionsjMethods to be Transferred to HPLC with HALO 60 Agilent 1100 QuaternaryShimadzu Prominence Agilent 1200SL Binary 00U

2.4

g y Agilent 1200 to Agilent 1100 and Shimadzu yMethods to be Transferred to HPLC with HALO 6 g Q y Agilent 1200SL Binary

20m

AU

60

Agilent 1200 to Agilent 1100 and Shimadzu34500 9 0 8

m

788

2.7

g g

189 30

.198 0.7

9

2

R l29500

0. 0.

216 bar 211 bar 212 bar 65

0

129

8 222.1 x 50 mm Results29500 216 bar 211 bar 212 bar .46 1. 868

272

.422.1 x 50 mm Results

24500

40

2 1 502 1 50 00

0. 2.8

3.21HALO C18

Di i t t t th t ff t th d24500

AU

2.1 x 50 mm2.1 x 50 mm 20AU 2 1 x 50 mm 10HALO C18

• Resolution results for respective peak pairs ranged from 97 120% Di d i t t t f t f f• Discuss important parameters that affect method y mA

9

. 500 4 L

2.1 x 50 mm0 4 L

2m

A 2.1 x 50 mm • Resolution results for respective peak pairs ranged from 97-120% • Discussed important parameters for transfer of an• Discuss important parameters that affect method ACN/W t M OH/W t19500si

ty 719 0.4 µL0.4 µL 76 0 5 µL 76

1

eso u o esu s o espec ve pe p s ged o 97 0%f h A il 1100 d f 81 108% f h Shi d P i

• Discussed important parameters for transfer of an p p ACN/Water MeOH/Water ens

612 0.

0.4 µL 0.4 µL

98 57 0.7 0.5 µL 1.7 for the Agilent 1100 and from 81-108% for the Shimadzu Prominence

p pACN viscosities 1 01 0 91 0 75 0 62 0 52 MeOH viscosities 1 62 1 47 1 21 1 00 0 83 14500nt

e

0 280 0.6

667

0.29

0.65 17 19

for the Agilent 1100, and from 81-108% for the Shimadzu Prominence.tr n f r f r i r ti nd r di nt m th d

ACN viscosities 1.01 0.91 0.75 0.62 0.52 MeOH viscosities 1.62 1.47 1.21 1.00 0.83 In 20 0.2

0.6

10 67

00.

7

00U 79 97 2.21

91

g ,i r ti nd r di nt m th d in HALOtransfer for isocratic and gradient methods ID Length ID Length 9500 52 5 0 0.

2

33 40 100

mA

U

7 0.97

1.69 2

2.29 • Injector program feature of Agilent 1100 system and software allowed isocratic and a gradient method using HALOtransfer for isocratic and gradient methods. ID

( )Length ( ) 25°C 30°C 40°C 50°C 60°C (mm)

e gt(mm) 25°C 30°C 40°C 50°C 60°C

9500

0.25 .225

1.77

082

996

1.33

1.94

271 0.0 1.0 2.0 3.0

1m

.777 0 1

1

2 • Injector program feature of Agilent 1100 system and software allowed isocratic and a gradient method using HALO g(mm) (mm) 25°C 30°C 40°C 50°C 60°C (mm) (mm) 25°C 30°C 40°C 50°C 60°C

4500

0

8

1 1

2.0

0.9 1

2.2

Time (min)0.

941 Injector program feature of Agilent 1100 system and software allowed

f dj f ff i d l l i i j i d lg g

3 0 20 3 12 3 46 4 20 5 00 5 00 3 0 20 1 93 2 15 2 60 3 10 3 10 4500

.528

Time (min)

1. for adjustment of effective delay volume using injection delay l i h 3 diff ID i l hD f f i i d di3.0 20 3.12 3.46 4.20 5.00 5.003 0 30 2 26 2 51 3 05 3 69 4 40

3.0 20 1.93 2.15 2.60 3.10 3.103 0 30 1 40 1 56 1 89 2 29 2 73 0

0 0

8 36 2 0 67 i i j d lfor adjustment of effective delay volume using injection delay. columns with 3 different IDs in same length• Demonstrate transfer of an isocratic and gradient 3.0 30 2.26 2.51 3.05 3.69 4.40 3.0 30 1.40 1.56 1.89 2.29 2.73 ‐500

0 0 1 0 2 0 458

2.0

2.41 0.67 min inj. delay

j y g j y columns with 3 different IDs in same length• Demonstrate transfer of an isocratic and gradient 3 0 50 1 46 1 62 1 97 2 38 2 84 3 0 50 0 91 1 00 1 22 1 48 1 76 0.0 0.5 1.0 1.5 2.0 2.5 3.00.0 1.0 2.0

Time (min) 0.0 1.0 2.0 0. 2 j y• Demonstrated changes in selecti it and resol tion on Agilent 1100

columns with 3 different IDs in same lengthDemonstrate transfer of an isocratic and gradient 3.0 50 1.46 1.62 1.97 2.38 2.84 3.0 50 0.91 1.00 1.22 1.48 1.763 0 75 0 63 0 69 0 85 1 02 1 24

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Ti ( i )

Time (min) Time (min)

625 • Demonstrated changes in selectivity and resolution on Agilent 1100

gg3.0 75 1.01 1.12 1.36 1.65 2.00 3.0 75 0.63 0.69 0.85 1.02 1.24 Time (min.)

257

301

374

833 2.6 Demonstrated changes in selectivity and resolution on Agilent 1100

h d f UHPLC HPLC d i 3 0 100 0 78 0 86 1 04 1 26 1 50 3 0 100 0 48 0 53 0 64 0 78 0 93 0.2

0.3

0.3

2.8

00

2

with different injection delay times P d l f h d fmethod from UHPLC to HPLC and summarize 3.0 100 0.78 0.86 1.04 1.26 1.50 3.0 100 0.48 0.53 0.64 0.78 0.93 265 bar 244 bar 245 bar 0 30

86 with different injection delay times. • Presented results from method transfermethod from UHPLC to HPLC, and summarize 3.0 150 0.53 0.59 0.71 0.86 1.00 3.0 150 0.33 0.37 0.44 0.53 0.62 40 40

265 bar 244 bar 245 bar0.0 1.0 2.0 3 .0

87

j y • Presented results from method transfermethod from UHPLC to HPLC, and summarize 3.0 150 0.53 0.59 0.71 0.86 1.00 3.0 150 0.33 0.37 0.44 0.53 0.6269500 4 0.0 1.0 2.0

Time (min) 8 553 3

157

• G di nt n n Shi dz t / inj ti n d l h d jPresented results from method transfer

72

( )

838

2.5 3.1 • Gradients run on Shimadzu system w/o injection delay showed major

lt 59500 4 0.6 7 1.

2 Gradients run on Shimadzu system w/o injection delay showed major i t d di d i th t iresults ACN/Water MeOH/Water 30

0.25

571

30 00AU

.61

801 changes in selectivity experiments and discussed issues that can ariseresults. ACN/Water MeOH/Water 49500

0

0.5

4 3 0 503 0 50

3 2m

A 1. 2.8 changes in selectivity. experiments, and discussed issues that can arise.

ACN viscosities 1 01 0 91 0 75 0 62 0 52 MeOH viscosities 1 62 1 47 1 21 1 00 0 8349500

AU

.624 3.0 x 50 mm3.0 x 50 mm AU 3.0 x 50 mm

m 2 g y p ,ACN viscosities 1.01 0.91 0.75 0.62 0.52 MeOH viscosities 1.62 1.47 1.21 1.00 0.83

39500y 0m

A 0

1 L1 L

m 3.0 x 50 mm

Sh i f i j i d l f diID Length ID Length 39500si

t 20 1 µL 1 µL 20 72 35 1 µL 13 97 79A il 1100 Q P id d id f f l f f• Show importance of injection delay for gradient (mm)g

(mm) 25°C 30°C 40°C 50°C 60°C (mm)g

(mm) 25°C 30°C 40°C 50°C 60°C en 231

µµ

0.27

1 0.73

1 µL

0 1.3

2.8 .27Agilent 1100 Quaternary • Provided guidance for successful transfer of• Show importance of injection delay for gradient (mm) (mm) 25°C 30°C 40°C 50°C 60°C (mm) (mm) 25°C 30°C 40°C 50°C 60°C 29500In

t 0.2

55 73 450

.621

800

100 1 2 3Agilent 1100 Quaternary

Ob i • Provided guidance for successful transfer ofShow importance of injection delay for gradient 2.1 20 1.53 1.70 2.06 2.45 2.45 2.1 20 0.95 1.05 1.28 1.52 1.52

I

0 9 1.15

1.67 72 0.24 0

0.68 1 Observations Provided guidance for successful transfer of p j y g 2.1 20 1.53 1.70 2.06 2.45 2.45

2 1 30 1 11 1 23 1 49 1 81 2 162.1 20 0.95 1.05 1.28 1.52 1.522 1 30 0 69 0 76 0 93 1 12 1 34 19500

1

209 1 1

1.9 10

0

2 6

Observations gh d f

2.1 30 1.11 1.23 1.49 1.81 2.16 2.1 30 0.69 0.76 0.93 1.12 1.34 19500

0.

3 6 1.27

2

.856 78

h d f UHPLC HPLC i HALOmethod transfer 2 1 50 0 72 0 79 0 97 1 17 1 39 2 1 50 0 44 0 49 0 60 0 72 0 86 9500 0.49 .946 1 1. 2.1

643 0 50 • Flow cell volume and path length differences among instruments methods from UHPLC to HPLC using HALOmethod transfer. 2.1 50 0.72 0.79 0.97 1.17 1.392 1 75 0 49 0 55 0 67 0 81 0 98

2.1 50 0.44 0.49 0.60 0.72 0.862 1 75 0 31 0 34 0 41 0 50 0 61

9500

0

0 0

1.9

0 93 i i j d l3.0 x 50 mm • Flow cell volume and path length differences among instruments methods from UHPLC to HPLC using HALOmethod transfer. 2.1 75 0.49 0.55 0.67 0.81 0.98 2.1 75 0.31 0.34 0.41 0.50 0.61

500

0 0 1

400 0.93 min inj. delayHALO C18

Flow cell volume and path length differences among instruments d k d h i h

methods from UHPLC to HPLC using HALO 2 1 100 0 38 0 42 0 51 0 62 0 74 2 1 100 0 24 0 26 0 32 0 38 0 46 ‐500

0 0 1 0 2 0 0 0 1 0 2 0 3 0

4 j yHALO C18 caused peak areas and heights to varyg

2.1 100 0.38 0.42 0.51 0.62 0.74 2.1 100 0.24 0.26 0.32 0.38 0.46 0.0 0.5 1.0 1.5 2.0 2.5 3.00.0 1.0 2.0

Time (min) 0.0 1.0 2.0Time (min)

0.0 1.0 2.0 3.0 caused peak areas and heights to vary.F d C l2.1 150 0.26 0.29 0.35 0.42 0.49 2.1 150 0.16 0.18 0.22 0.26 0.30 Time (min )

( ) Time (min) Time (min)

0 2

p g yFused-Core columns2.1 150 0.26 0.29 0.35 0.42 0.49 Time (min.)

0 830

632

9 1 34 i i j d l • Anal tical a elength band idth and slit idth on Agilent 1200 DAD Fused-Core columns.

60300

1.8

3 581 2.6

079 1.34 min inj. delay • Analytical wavelength bandwidth and slit width on Agilent 1200 DAD

40 0335 bar 282 bar 284 bar 00 2.1

2463

983

2.5 3.0 j y Analytical wavelength bandwidth and slit width on Agilent 1200 DAD

• Calculations are approximate 99000 4 40335 bar 282 bar 284 bar 40

22.

2

659

673

AU

1.9 2

had to be adjusted lower to give comparable peak areas• Calculations are approximate. 89000

2

2.6

2.6

mA

12 had to be adjusted lower to give comparable peak areas.Ca cu a o s a e app o a e. 89000

53

2 2

00m

2.81

j g p p79000 30 0.

25

706

4 6 504 6 50

0 U

0320 2

P f i fl f 80% f 400 b 69000

3 0

7

0.7 4.6 x 50 mm4.6 x 50 mm 30 4 6 x 50 mm m

AU

3.10 852 3• Pressure for maximum flow rate set for 80% of 400 bar 69000

AU

597

2 L2 L AU

4.6 x 50 mm m 33.

18

902

153• Pressure for maximum flow rate set for 80% of 400 bar. 59000ty m

A 0.65

4 2 µL 2 µL mA

2 µL 868 3

85 2.9 3.1

70

49000

59000

nsit 20 6 7 0.6 µ2 µL

20 271 2 µL

00 2.800 1.58 .27

49000en .216

.227 2

0.2

53

20 21 1 3

• E ti t d ith 0 005” ID t bin in t pi l l n th ( 60 70 ) t nt 39000Int 0 0

243

33 0.75 45 9433 0• Estimates made with 0.005” ID tubing in typical length (~60-70 cm) to account 39000I

0 37 08

0.2

0.63

693 0

990

2.94 .29

392

2.03

060Estimates made with 0.005 ID tubing in typical length ( 60 70 cm) to account

29000 10

204

1.23

1.80

132

10 0.6

1.9 2 3

1.3 2

2.0

f t ( t i l di fl ll d t l ) 19000 0.2 1 1

2.1

314

23 9

10 1

for system pressure (not including flow cell and autosampler) 19000

509 1.3

1.92

2.24

9

901 0 2 0 3 0for system pressure (not including flow cell and autosampler)9000 0

0.5 2

1.791.0 2.0 3.0

Ti ( i )1000

0 0 0

1Time (min)‐1000

0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 1.0 2.0Time (min)

0.0 1.0 2.0 0.0 1.0 2.0 3.0

Time (min )Time (min) Time (min) Time (min)Time (min.) ( )

R l f I i M h d T f f G di T f A il 1100 H Sh ld O MI ti M th d T f E l Results for Isocratic Method Transfer from Gradient Transfer to Agilent 1100 How Should One MeasureG l (U)HPLC M h d T f C Isocratic Method Transfer Example Results for Isocratic Method Transfer from Gradient Transfer to Agilent 1100 How Should One MeasureGeneral (U)HPLC Method Transfer Concerns Isocratic Method Transfer Example Results for Isocratic Method Transfer from Gradient Transfer to Agilent 1100 How Should One Measure General (U)HPLC Method Transfer Concerns d p gGeneral (U)HPLC Method Transfer Concerns

T f h d A il 1100 d 1200SL S Shi d t A il t 1100 d 1200 ith Diff t HALO C l ID h d T f S ?( )

Transfer method to Agilent 1100 and 1200SL Systems Shimadzu to Agilent 1100 and 1200 with Different HALO Column IDs Method Tr nsfer S ccess?Transfer method to Agilent 1100 and 1200SL Systems Shimadzu to Agilent 1100 and 1200 with Different HALO Column IDs Method Transfer Success?g y d u o g e 00 d 00 e e o u s Method Transfer Success? O i i l Shi d I M h d R lACN/W t Original Shimadzu Inst Method Results

• C l mn l n th nd di m t r ACN/Water Original Shimadzu Inst. Method Results Agilent 1200SL Agilent 1100 QuaternaryAgilent 1200SL Agilent 1100 Quaternary• Column length and diameter ACN i iti 1 01 0 91 0 75 0 62 0 52

gd %

g0 42 mL/min 1 µL inj

g y0 86 mL/min 2 µL inj

g0 42 mL/min 1 µL inj

g y0 86 mL/min 2 µL inj • Comp r ble pe k sh pe efficienc selecti itColumn length and diameter ACN viscosities 1.01 0.91 0.75 0.62 0.52 • Resolution results for respective peak pairs ranged from 91 117% 2 1 50 HALO C18

0.42 mL/min, 1 µL inj.N I j ti D l

0.86 mL/min, 2 µL inj.I j ti D l 0 93 i3 0 50 HALO C182 1 50 HALO C18

0.42 mL/min, 1 µL inj.N I j ti D l

0.86 mL/min, 2 µL inj.I j ti D l 0 93 i3 0 50 HALO C183 0 50 HALO C18 • Comparable peak shape efficiency selectivity

I b d d d l ID Length • Resolution results for respective peak pairs ranged from 91-117% 2.1 50 mm HALO C18 No Injection Delay Injection Delay 0.93 min.3.0 50 mm HALO C18002.1 50 mm HALO C18 No Injection Delay Injection Delay 0.93 min.3.0 50 mm HALO C1800 3.0 50 mm HALO C1800 Comparable peak shape, efficiency, selectivity, • Instrument brand and model ID Length Column: 4 6 x 50 mm HALO C18

p p p gf h A il 1100 d f 95 107% f h A il 1200 60 5460 5460 54

p p p , y, y,Instrument brand and model (mm) (mm) 25°C 30°C 40°C 50°C 60°C Column: 4.6 x 50 mm, HALO C18 for the Agilent 1100 and from 95-107% for the Agilent 1200 0 22

6

350 22

6

35

6

35

d l i(mm) (mm) 25 C 30 C 40 C 50 C 60 CM bil Ph 43 57 A/B

for the Agilent 1100, and from 95-107% for the Agilent 1200.

200

42 2.3

200

42 2.3

2.3

and resolution• Instrument pressure limit 3.0 20 3 12 3 46 4 20 5 00 5 00 Mobile Phase: 43:57 A/Bg , g

2 1.4 22 1.4 22 and resolution• Instrument pressure limit 3.0 20 3.12 3.46 4.20 5.00 5.00

3 0 30 2 26 2 51 3 05 3 69 4 40/

• Theoretical plate counts increased with k for respective analytes as

1

1 0

1

1 00

and resolutionp3.0 30 2.26 2.51 3.05 3.69 4.40 • Theoretical plate counts increased with k for respective analytes as 61 0061 0000

• P pi t t p l p 3 0 50 1 46 1 62 1 97 2 38 2 84p p y

d k 8 9 U .76

9 40 30 32 9U .76

9 40 30 32 940 30 32 9• Pumping system type: low pressure 3.0 50 1.46 1.62 1.97 2.38 2.84 Mobile Phase A: 0 020M sodium phosphate expected up to ~k = 8-9 AU 9 7 1. 19 1

4U

83 3 81 63 79AU 9 7 1. 19 1

4U

83 3 81 63 79

4U

83 3 81 63 79 C bl i j i i j i biliPumping system type: low pressure 3 0 75 1 01 1 12 1 36 1 65 2 00 Mobile Phase A: 0.020M sodium phosphate expected, up to k = 8-9 mA 7 979

69 .2 291

AU

1.8

983 58 2.6

.07

mA 7 979

69 .2 291

AU

1.8

983 58 2.6

.07

AU

1.8

983 58 2.6

.07 • Comparable injection to injection repeatabilitymixing vs high pressure mixing3.0 75 1.01 1.12 1.36 1.65 2.00 p p

(pH=2 5)p , p

00m

777

0.9 .6 1 2 .2 mA 1 .9 2. 2

12 3.00m

777

0.9 .6 1 2 .2 mA 1 .9 2. 2

12 3.mA 1 .9 2. 2

12 3. • Comparable injection-to-injection repeatabilitymixing vs. high pressure mixing 3.0 100 0.78 0.86 1.04 1.26 1.50 (pH=2.5) • Plate counts increased slightly with increasing ID (4 6 > 3 0 > 2 1) 10 0.7 0 1 94 2

0m 1 2

8110 0.7 0 1 94 2

0m 1 2

810m 1 2

81

Comparable injection to injection repeatabilityg g p g 3.0 100 0.78 0.86 1.04 1.26 1.503 0 150 0 53 0 59 0 71 0 86 1 00 Mobil Ph B: 50:50 M OH/ACN pr mi • Plate counts increased slightly with increasing ID (4.6 > 3.0 > 2.1). 1

8 0 1.9

36 2 00 2.8

02 53

1

8 0 1.9

36 2 00 2.8

02 5300 2.8

02 53

p p yG di t d l l d fl 3.0 150 0.53 0.59 0.71 0.86 1.00 Mobile Phase B: 50:50 MeOH/ACN premix a e cou s c eased s g y w c eas g (4.6 3.0 . ).

58 1 03 412 20 85

290 15 7058 1 03 41

2 20 85

290 15 7020 85

290 15 70 /• Gradient delay volume and flow

/ p

.45

2.0

2.4 58 2.9

3.1

27.45

2.0

2.4 58 2.9

3.1

2758 2.9

3.1

27 • C p bl S/N R ti (i p t n LOD ndGradient delay volume and flow

74 0. 2 2 2 1.5

33 0

2 33.

2

74 0. 2 2 2 1.5

33 0

2 33.

2

2 1.5

33 0

2 33.

2 • Comparable S/N Ratio (impact on LOD andrate/pressure dependence ACN/Water 37 392 1

03 060 3

37 392 1

03 060 3

392 1

03 060 3 Comparable S/N Ratio (impact on LOD and rate/pressure dependence ACN/Water Flow Rate: 3 0 mL/min Ob i 0.

3 .3 2.0

2.00.3 .3 2.0

2.0.3 2.0

2.0 p / ( p/p p

ACN viscosities 1 01 0 91 0 75 0 62 0 52Flow Rate: 3.0 mL/min. Observations 0 0

0 1 2 20 0

0 1 2 20 1 2 2

LOQ li i UV l li )Mi lACN viscosities 1.01 0.91 0.75 0.62 0.52

P 338 BObservations 0 00 00

LOQ linearity UV spectral quality etc )• Mixer volume ID Length Pressure: 338 Bar 0 0 1 0 2 0 2 0 3 00 0 1 0 2 0 2 0 3 02 0 3 0 LOQ, linearity, UV spectral quality, etc.)Mixer volume ID Length T ° • DAD reference signal had to be turned off to avoid RI disturbance near 0.0 1.0 2.0 2.0 3.00.0 1.0 2.0 2.0 3.02.0 3.0 LOQ, linearity, UV spectral quality, etc.)

(mm) (mm) 25°C 30°C 40°C 50°C 60°C Temperature: 35°C • DAD reference signal had to be turned off to avoid RI disturbance near Time (min) Time (min)Time (min) Time (min)Time (min)Q, y, p q y, )

• Flow Rate(mm) (mm) 25 C 30 C 40 C 50 C 60 C2 1 20 1 53 1 70 2 06 2 45 2 45

Temperature: 35 C DAD reference signal had to be turned off to avoid RI disturbance near Time (min) Time (min)Time (min) Time (min)Time (min)• Flow Rate 2.1 20 1.53 1.70 2.06 2.45 2.45 Detection: UV 254 nm VWD start of run A il t 1100 Q tA il t 1100 Q t A il t 1100 Q tA il t 1100 Q t C bl d i i d h2 1 30 1 11 1 23 1 49 1 81 2 16Detection: UV 254 nm, VWD start of run. Agilent 1100 QuaternaryAgilent 1100 Quaternary2 1 50 mm HALO C18 4.6 50 mm HALO C18 Agilent 1100 QuaternaryAgilent 1100 Quaternary2 1 50 mm HALO C18 4.6 50 mm HALO C184.6 50 mm HALO C18 • Comparable accuracy and precision and other• Column Oven type and Column 2.1 30 1.11 1.23 1.49 1.81 2.16

,I j i V l 2 0 L

g y2.0 mL/min, 4.8 µL inj.0 9

g y0.42 mL/min, 1 µL inj.

2.1 50 mm HALO C18 4.6 50 mm HALO C18 g y2.0 mL/min, 4.8 µL inj.0 9

g y0.42 mL/min, 1 µL inj.

2.1 50 mm HALO C18 4.6 50 mm HALO C184.6 50 mm HALO C18 • Comparable accuracy and precision and other• Column Oven type and Column 2 1 50 0 72 0 79 0 97 1 17 1 39 Injection Volume: 2.0 µL F i ti ti fl t d i j ti l • D t r t f r A il nt 1200 t t 40 Hz t m r l l m t h 50 Hz2.0 mL/min, 4.8 µL inj.Injection Delay 0 41 min00 59

0.42 mL/min, 1 µL inj.Injection Delay 1 74 min

2.0 mL/min, 4.8 µL inj.Injection Delay 0 41 min00 59

0.42 mL/min, 1 µL inj.Injection Delay 1 74 min

Comparable accuracy and precision and other Co u Ove ype d Co u 2.1 50 0.72 0.79 0.97 1.17 1.39 Injection Volume: 2.0 µL For isocratic separation, flow rate and injection volume are • Data rate for Agilent 1200 was set at 40 Hz to more closely match 50 Hz Injection Delay 0.41 min.4 55 Injection Delay 1.74 min.

75

Injection Delay 0.41 min.4 55 Injection Delay 1.74 min.

7575

p y pTemperature 2.1 75 0.49 0.55 0.67 0.81 0.98 Sample Solvent: 50:50 MeOH/water

p , jl d ith ti f l ID d d b l ti

Data rate for Agilent 1200 was set at 40 Hz to more closely match 50 Hz

3. 0 673. 0 670 67 fi f itTemperature 2.1 75 0.49 0.55 0.67 0.81 0.982 1 100 0 38 0 42 0 51 0 62 0 74

Sample Solvent: 50:50 MeOH/water scaled with ratio of column IDs squared and by volume ratio, rate of Shimadzu

3

600

1.63

600

1.6

600

1.6 figures of meritp

2.1 100 0.38 0.42 0.51 0.62 0.74 Data Rate: 50 Hzq y ,

i lrate of Shimadzu. 6 16 16 1 figures of merit

• D t t r fl ll nd l m0 38 0 0 5 0 6 0

2 1 150 0 26 0 29 0 35 0 42 0 49Data Rate: 50 Hz. respectively 7 77 7

g• Detector flow cell and volume 2.1 150 0.26 0.29 0.35 0.42 0.49

R Ti 0 02p y

Fl ll th l th diff i t t d k

75 957

417 19

75 957

417 19 19Detector flow cell and volume Response Time: 0.02 sec. • Flow cell path length differences among instruments caused peak areas U

87 3.9

4.4 0U 91 5U

87 3.9

4.4 0U 91 50U 91 5 il id i l i i !D d d i

Response Time: 0.02 sec. Flow cell path length differences among instruments caused peak areas

00AU

1 3.8 3

28

4 400

AU 0 1.9

75 100AU

1 3.8 3

28

4 400

AU 0 1.9

75 1400

AU 0 1.9

75 1 • Not necessarily identical retention times!• Detector data rate and response time Flow Cell: 2 5 µL semi-micro 2 and heights to vary 20m

A

80 51 3

12 4m

A

2 881 37 5120mA

80 51 3

12 4m

A

2 881 37 514

mA

2 881 37 51 • Not necessarily identical retention times!Detector data rate and response time Flow Cell: 2.5 µL semi-micro 2

ID and heights to vary. 2m 78 .0 4.1

7 4 m 32 1.8

13 2.3

.42m 78 .0 4.1

7 4 m 32 1.8

13 2.3

.4m 32 1.8

13 2.3

.4 Not necessarily identical retention times! pLC S t m Shim dz Pr min n

ID a d e g ts to va y.

2 2.7 3. 4 27 484

8 03 1 11

2 22 2.7 3. 4 27 484

8 03 1 11

2 203 1 11

2 2

y• Injector type and injection volume LC System: Shimadzu Prominence 2

colIDFF A l ti l l th b d idth d lit idth A il t 1200 DAD 32

2 .22

4.4 08 00 1. 2.1

00 6932

2 .22

4.4 08 00 1. 2.1

00 6900 1. 2.1

00 69• Injector type and injection volume yUFLC XR

212 col

ll FF • Analytical wavelength bandwidth and slit width on Agilent 1200 DAD 43 4. 4 .60

20

2 20 5643 4. 4 .60

20

2 20 5620

2 20 56j yp j UFLC XR 12

colcol IDFF Analytical wavelength bandwidth and slit width on Agilent 1200 DAD 9 2. 27 55 5 4. 2 2.

2

2.59 2. 27 55 5 4. 2 2.2

2.52 2.2

2.5

P i 600 b12

colcol IDy g g

had to be adjusted lower to give comparable peak areas 239

22 25 455 2 2

239

22 25 455 2 22 2

Pressure maximum: 600 bar 1 colID had to be adjusted lower to give comparable peak areas. 2.2 3.2

3.2 .42.2 3.2

3.2 .4Pressure maximum: 600 bar 1 col had to be adjusted lower to give comparable peak areas.

0 2 3 3 3 00 2 3 3 3 00

2

0 00 00

2 2 0 3 0 4 0 5 02 0 3 0 4 0 5 0

2

LID 2.0 3.0 4.0 5.0 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.62.0 3.0 4.0 5.0 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.60.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

22 ll LID

Time (min)

0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Time (min)Time (min)

0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Time (min)

0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6Time (min) 22 colcol LIDVV

Time (min) Time (min)Time (min) Time (min)Time (min) 22 colcol

i ji j VV 12 colinjcolinj LID

VV

3 70% ACN/water w/0 1% HCOOH in 2 7 min3 70% ACN/water w/0 1% HCOOH in 2 7 minPeak identities (in order): hydroquinone, resorcinol, catechol, phenol, 4-nitrophenol, 4,4’-biphenol, 2-chlorophenol, 4-chlorophenol, 2,2’-biphenol, 2,6 –dichlorophenol,

12 coljcolj LID

3–70% ACN/water w/0.1% HCOOH in 2.7 min.3–70% ACN/water w/0.1% HCOOH in 2.7 min.Peak identities (in order): hydroquinone, resorcinol, catechol, phenol, 4 nitrophenol, 4,4 biphenol, 2 chlorophenol, 4 chlorophenol, 2,2 biphenol, 2,6 dichlorophenol,

2 4 di hl r ph n l11 colcol LID 2,4-dichlorophenol11 colcol