Optimizing Selectivity for Preparative Separations: … · Optimizing Selectivity for Preparative...

Optimizing Selectivity for Preparative Separations: Mixed-Mode Chromatographyversus Reversed Phase and Hydrophilic Interaction Chromatography

METHODS METHODS ((cont’dcont’d))

Maximum separation (α > 1.2).Short retention (k’ < 4).High solubility in eluent.Elution order.Volatile Mobile Phase.Preferably isocratic conditions for separation.

METHODS METHODS ((cont’dcont’d)) METHODS METHODS ((cont’dcont’d)) METHODS METHODS ((cont’dcont’d))

Mixed-Mode Chromatography Columns:

1. Allow preparative purification of molecules not possible in the past.

2. Allow automation by providing separation of both hydrophilic and hydrophobic compounds in one run under isocratic conditions.

3. Allow controllable retention/separation by multidimensionalmethod development.

CONCLUSIONSCONCLUSIONS

Vera LeshchinskayaBristol-Myers Squibb Co., PO Box 4000 Princeton, NJ 08543-4000 USA,

Tel: (609)252-3935, E-mail: [email protected]

Ming ZengPfizer Inc., 185N Eastern Point Road, Groton, CT 06340

Tel: (860)686 1044, E-mail: [email protected]

METHODSMETHODS

Criteria for Evaluating Preparative Methods

Column Selection for Screening System

Carboxylic/aminoEmbedded basic/ acidic group

Mixed-Mode Chromatography Columns

Can be operated in HILIC modeDiol, Amino-columnsPolar Bounded Phases

Inorganic-organic hybrid particlesHigh pH resistant

Ether, amide, carbamateEmbedded polar group

Hydrophilic endcapping, shortalkyl chains, wide pore diameter,

“Aqua” columns

Low surface coverageEnhanced polar selectivity

Phenyl(“pi-acidic”,“pi-basic”), cyanoAlternative Selectivity

C8Less Hydrophobic

C18Moderately Hydrophobic

High carbon load, C30Highly hydrophobic

Reversed Phase

Functional groupCategory

Mixed-Mode Primesep Columns

B

B

B

A

A

A

Optimizing the Separation MethodDevelopment for Polar Compounds

The desiredAre highly basic ionic compounds;Are submitted in the form of HCl salt;Do not elute under normal phase conditions and show little retention underreverse phase conditions;Contain closely related impurities;Contain highly retentive reagent (~40% by weight).

Objective for preparative purification:Impurities, as measured by HPLC, should not exceed 1% each;The yield of the desired material should be > 90%;Purity should be at least 98%.

Newcolumn

Usedcolumn

ODS AQ ColumnStability under Usage at

0.1% TFA, pH = 1.9Modifiers Comparison on

YMC ODS AQ Media

pH=2.6

pH=2.3

Pilot Run to Investigate the Possibility for External Recycle

0 10 20

fraction #

pea

k ar

ea

imp.1

imp.2

desired

0 20 40

fraction #

pea

k ar

ea

imp.1

imp.2

desired

initial injection(50:1 load ratio)

recycle of fractions#7 & 8

Data Comparison for Hydrophobic, HILIC andMixed-Mode Type of Chromatography

302520151050

ODS AQ, 10 um, 4.6x25mm, 100/0.1 H20/TFA

AA

Product Bis-adduct

Hydrophobic Chromatography

Bakerbond DIOL, 5 um, 4.6x25mm, 70/30/0.1 ACN/H20/TFA

109876543210Retention Time (min)

Product AA

Bis-Adduct

HILIC Chromatography

Primesep 200, 5 um, 4.6x25mm, 20/80/0.1 ACN/H20/TFA

Primesep 200, 5 um, 4.6x25mm, 80/20/0.1 ACN/H20/TFA

35302520151050

Product Bis-adduct

AA

Mixed-Mode Chromatography

50454035302520151050

Product Bis-adduct

AA

Sample Profile on ODS AQ and Primesep 200 Column

05

1015202530354045

0/100

/0.1

20/8

0/0.1

40

/60/0

.1

60/4

0/0.1

80

/20/0

.1

ACN/H2O/TFA

Tr, m

in

ace tamidineP RIMESEPace tamidine ODS AQ

P ro duct ODS AQ

P ro duct P RIMESEP

Bis -adduc t ODS AQ

Bis -adduc tP RIMESEP

What is ChangedImpurity profile.Stationary phase technology available on the market.

ObjectiveFind conditions that would allow to obtain 98% pure desired.Develop efficient process using 10 µ media, automated injections.

New Sample Profile (ODS AQ Column)ODS AQ, 10 um, 4.6x250 mm, 3/97/0.1 ACN/H20/TFA for 12 min, then

graduent to 50/50/0.1 ACN/H2O/TFA




NaCl, ELSD

Sample, UV, 210 nm

ZnAc, ELSD

Method Development by HILIC Chromatography


27742036.001535698.50 582529.69

Bakerbond Diol, 5 um, 4.6x250 mm, 80/20/0.1 ACN/H20/TFA

YMC Diol, 5 um, 4.6x250 mm, 90/10/0.1 ACN/H2O/TFA


NaCl, ELSD

ZnAc, ELSD

Sample, ELSD

NaCl, ELSD

ZnAc, ELSD

Sample, ELSD

Method Development by Mixed-Mode ChromatographyPrimesep 100, 10 um, 4.6x250 mm, 50/50/0.3 ACN/H2O/TFA

NaCl, ELSD




ZnAc, ELSD

Sample, ELSD

Impurity Profile on ODS AQ and Primesep 100 Columns

300250200150100500

30025020015010050

ODS AQ. 10 um, 1 ml/min, 3/97/0.1 ACN/H2O/TFA,

Primesep 100 10 um 1ml/min 50/50/0.3 ACN/H2O/TFA


108278272.0010668928.00 3565569.001425165.50

Loading Study for HCI Salt on YMC ODS AQ Column O D S A Q , 1 0 u m , 1 m l/m in , 2 4 0 n m , s a m p le d is s o lv e d in 1 0 0 /0 .1

H 2 O /T F A a t 1 0 0 m g /m l3 /9 7 /0 .1 A C N /H 2 O /T F A fo r 1 2 m in , th e n g ra d u e n t to 5 0 /5 0 /0 .1

A C N /H 2 0 /T F A



A n a ly tic a l in je c tio n

2 m g

4 m g

6 m g

1 0 m g

1 5 m g

Loading Study for HCI Salt on Primesep 100 Column

HCL SALT, 7 MG ON PRIMESEP 100 COLUMN

0

5000

10000

15000

20000

1 2 3 4 5 6 7 8 9 10

FR#

Are

a(m

AU

*s)

Reproduction ofpreparative loading study

Primesep 100, 10 um, 1ml/min, 230 nm

50/50/0.3 ACN/H2O/TFA, 7 mg/per injection


HPLC Method Development for FourClosely Structure-Related Compounds

Objective: develop an analytical method suitable for sample analysis/puritycheck and preparative scale-up

Comment: the sample contains carboxylic functionality in the side chain

R1

R2

R1

R3

R2

R3

R3

R2

R1

R1

R2

R3

Results of HPLC Screening for FourClosely Structure-Related Compounds (1)

ODS AQ, 10 um


1050Retention Time (min)ACN/H2O/TFA

60/40/0.1

MeOH/H2O/TFA 80/20/0.1


ACN/H2O/TFA 70/30/0.1

SYMMETRY SHIELD, 5 um



KLASSIX C10-CN, 5 um


ACN/H2O/TFA 60/40/0.1


MeOH/H2O/TFA80/20/0.1


ZORBAX SB PHENYL, 5 um

151050Retention Time (min)MeOH/H2O/TFA

80/20/0.1

ACN/H2O/TFA 60/40/0.1



ACN/H2O/TFA 70/30/0.1

ES INDUSTRIES, FSP, 5 um



ACN/H2O/TFA 70/30/0.1



YMC J'SPHERE, 5 um

(2)


ACN /H 2O /TFA 60/40/0 .1


M eO H /H 2O /TFA 80/20/0.1


IP A /M eO H/H 2O /TFA 10/60/30/0.1


IPA /ACN /H2O /TFA 20/40/40/0.1

α 1 = 1 .14

a2 = 1 .19

a3 = 1 .07

α 1 = 1 .07

a2 = 1 .51

a3 = 1 .04

α 1 = 1 .05

a2 = 1 .47

a3 = 1 .05

α 1 = 1.09

a2 = 1.27

AB Z+PLU S, Supelco , 5 um

(3)


A C N /H 2O /H C O O H 60/40 /0 .9


A C N /H 2O /H 2S O 4 60/40/0 .02


A C N /H 2O /TF A 50/50/0 .05

P R IM E S E P B , ALL S E P , 5 u m


A C N /H 2O /H C O O H 70/30/0 .9

α 1 = 1 .14

a2 = 1 .34

a3 = 1 .10

α 1 = 1 .16

a2 = 1 .04

a3 = 1 .13

α 1 = 1 .24

a2 = 1 .19

a3 = 1 .19

α 1 = 1 .20

a2 = 1 .21

a3 = 1 .17

04-077

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