purospher star lp_application guide

7/25/2019 Purospher STAR LP_Application Guide

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Merck Millipore is a division of Merck KGaA, Darmstadt, Germanyhttp://www.merckmillipore.com/[email protected]


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Introduction

Purospher HPLC columns are based upon a high-purity, metal free silica forexcellent separations with good symmetry. The base material for Purospher high-purity HPLC columns is made from tetra-alkoxysilane. Due to the absence of metalsin the silica matrix and in combination with an excellent coverage of the silicasurface, this stationary phase enables tailing free chromatography of acidic, basic

and chelating compounds. This is of particular advantage for method development.The newest addition to the Purospher family is Purospher STAR LP RP-18endcapped. The packing material for Purospher STAR LP RP-18 endcapped hasbeen optimized on pressure which gives at least 25% less back pressure incomparison with existing Purospher STAR RP-18 endcapped. This will give moreopportunity to develop method for formulated drugs.

e app ca on gu e w us ra e eren app ca ons w exce en separa onon formulated product like Tablet, Capsules, Injections etc.


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Specification for Purospher STAR LP RP-18 endcapped

Sorbent: High purity silica gelParticle Size: 5mPore Size: 120 Surface Area: 330 m2/gmCarbon Load: 17 %pH range: 1.5 to 10.5Performance: > 70,000 N/m

Purospher STAR LP RP-18e Vs Competition

40

60

80

100

120

140

160

180

Backpressure

(Bar)

Back Pressure Comparison with MeOH/H2O 50:50 (v/v)


03

Instrument 1 Instrument 2

Purospher STAR LP250x4.6 mm, 5 m

179 164

Competition Column250x4.6 mm, 5 m

165 157

0


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Application Index

Formulation Name Compound class Therapeutic use AnalysisMethods

Atorvastatin Tablet Statins Lowering bloodcholesterol

IP 5

Chlorocresol, Beclomethasone,Miconazole Cream

Disinfectant Inflammatory skindisorder

In-house 6

Bisoprolol, Amlodipine Tablet Beta blocker & Cardiovascular In-house 7

Ciprofloxacin Eye drops Antibiotic Eye infection USP/IP 8

Cyanocobalamine Injection Vitamin B12 In-house 9

Fenofibrate Capsule Fibrate Reduce cholesterol &triglyceride

USP 10

Folic acid tablet Vitamin B9 IP 11

Hydrochlorothiazide, RamiprilTablet

Antihypertensive High blood pressure In-house 12

Ivermectin Injection Antiparasitic River blindness USP 13

Levofloxacin Tablet Antibiotic Bacterial infection IP 14

Mecobalamine Injection Vitamin JP 15

Metformin, Pioglitazole Tablet Antidiabetic High blood sugar In-house 16

Nebivolol Tablet Antihypertensive Cardiovasculardisease

IP 17


04

Ofloxacin Tablet Antibiotic Bacterial infection IP 18

Olanzapine Tablet Antipsychotic Bipolar disorder IP 19

Olmesartan, HydrochlorothiazideTablet

Angiotensin IIreceptor &Antihypertensive

Cardiovasculardisease

In-house 20

Olmesartan, Amlodipine Tablet Angiotensin IIreceptor &Antihypertensive


In-house 21

Olmesratan Tablet Angiotensin IIreceptor


In-house 22


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Purospher STAR LP Atorvastatin Tablet

Chromatographic Conditions

Column: Purospher STAR LP RP18e 250x4.6 mm, 5.0 m

Injection: 0 L

Detection: Shimadzu 2010 CHT, UV 246 nm

Cell: 10 L

Gradient: See table

Mobile Phase (v/v): : Acetonitrile : Tetrahydrofuran (92.5:7.5 v/v)

Time(min)

Flow(ml/min)

%B %C

0.0 1.8 100 0

20.0 1.8 100 0

35.0 1.5 25 75

40.0 1.5 25 75

55.0 1.5 100 0

200

400

600

800

1000

1200

Intensity(mV)

Atorvastatin

B: 5.75 g of ammonium dihydogen phosphate in 1L water.

Mix Buffer & Mobile phase A 58:42 (v/v).

C: Buffer:Mobile phase A & Methanol 20:20:60 (v/v)

emperature: 5 C

Diluent: cetonitrile: Water 40:60 (v/v)

ample: Crush 20 tablet. Weigh equivalent to 50 mg. of Atorvastatin. Disperse in 10 ml Methanol.

dd 20 ml Diluent: & sonicate for 30 min. Dilute to 100 ml & filter through 0.45 m filter paper.

Pressure Drop: 20 Bar to 200 Bar (3190 psi to 2900 psi)

. .

70.0 1.8 100 0

Chromatographic Data

No. Compound Time (min) Tailing Factor Theoretical Plate

1 Atorvastatin 19.6 1.0 11033

0

0 10 20 30 40 50

Retention Time (min)


05


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Purospher STAR LP Beclomethasone, Chlorocresol, Miconazole


Column: Purospher STAR LP RP-18e 250x4.6 mm, 5 mInjection: 20 LDetection: Shimadzu Prominence, UV 240nmCell: 10 LFlow Rate: 1.0 mL/minMobile Phase (v/v): 6.8 g of Sodium acetate trihydrate in 1 L water. Adjust pH to 7.4 with dilute H2SO4

Mix Acetonitrile and Buffer 70:30 (v/v)Temperature: 30 CDiluent: Methanol

0

40

80

120

160

200

Intensity(mV)

Miconazole

Beclomethas

one

Chlorocresol

Standard: Solution I: 50 mg Beclomethasone in 50 ml methanol

Solution II: 40 mg Chlorocresol in 100 ml volumetric flask. Add 35 ml methanol to dissolve.Add 10 ml Solution I & make up the volume with methanol.

Solution III:40 mg Miconazole in 100 ml volumetric flask. Add 35 ml methanol to dissolve.Add 5 ml Solution II & make up the volume with methanol.

Sample: 2 g of cream in 100 ml volumetric flask. Add 35 ml of methanol & sonicate for 15 min.Dilute to volume with methanol & filter through 0.45 m filter paper.

Pressure Drop: 46 Bar (667 psi)

0 5 10 15 20 25




1 Chlorocresol 4.5 1.2 12210

2 Beclomethasone 8.6 1.1 13911

3 Miconazole 18.7 1.2 16398


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Purospher STAR LP Ciprofloxacin Eye drops


Column: Purospher STAR LP RP-18e 250x4.6 mm, 5 m

Injection: 0 L

Detection: Shimadzu Prominence, UV 280 nm

Cell: 10 L

Flow Rate: 1.5 mL/min

Mobile Phase (v/v): 1.7 g of tetra-butyl ammonium phosphate in 1L water. Adjust pH to 2.0

ith ortho-phosphoric acid. Mix methanol & buffer 1:3 (v/v)

emperature: 30 C

Diluent: ater

tandard: 0.14 mg/ml in water

ample: 12 mg equivalent of ciprofloxacin from eye drop in 100 ml water.


10

20

30

40

50

Intensity(mV)

Ethylenediamneanalog

Ciprofloxacin

0 5 10 15 20



No. Compound Time (min) RRT Theoretical Plate

1 Ethylenediamine analog 5.8 0.8 6095

2 Ciprofloxacin 7.4 1.0 5090


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Purospher STAR LP Cyanocobalamine Injection



Injection: 50 L


Cell: 10 L


Mobile Phase (v/v): : 2.72 g of KH2PO4 in 800 ml water. Add 3.76 g hexane

Time

(min)

% A %B

0.0 80 20

17.0 80 20

17.1 40 60

30.0 40 60

30.1 80 20

40

60

80

100

120

140

Intensity(mV)

Cyanocobalami

ne

.

B: MethanolGradient Programme: See table

Temperature: 0 C

Diluent: ater

Standard: eigh 40 mg cyanocobalamine and dilute to 100 ml with water.

Pipette out 5 ml above solution dilute to 50 ml with water

Sample: Mix 10 injection ampoules. Dilute 3 ml of this solution to 25 ml with water.


40.0 80 20

0

0 8 16 24 32 40




1 Cyanocobalamine 14.5 1.1 6703


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Purospher STAR LP Fenofibrate Tablet



Injection: 0 L

Detection: Shimadzu Prominence, UV 285nm

Cell: 10 L


Mobile Phase (v/v): 136 mg potassium di-hydrogen phosphate in 1 L water. Adjust pH to 2.9 with

rtho-phosphoric acid. Mix methanol and buffer 4:1 (v/v)

50

100

150

200

250

Intensity(mV)

Fenofibrate

emperature: 30 C

Diluent: Mobile phase

tandard: 67 ppm of fenofibrate in mobile phase.

ample: Crush 20 capsules. Weigh equivalent to 67 mg of fenofibrate in 100 ml volumetric

lask. Add 80 ml of mobile phase. Sonicate for 25 min. Dilute 10 ml of the

solution to 100 ml with mobile phase and filter through 0.45 m filter paper.




1 Fenofibrate 9.2 1.0 8260

0

0 5 10 15 20



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Purospher STAR LP Folic acid tablet



Injection: 0 L


Cell: 10 L


Mobile Phase (v/v): 9.38 g of sodium perchlorate & 0.75 g of KH2PO4 in 900 ml water

djust pH to 7.2 with 0.1 M KOH & dilute to 1000 ml with water

Mix Buffer and Methanol 800:135 (v/v).

emperature: 30 C

Diluent: 0.57 % K2HPO4 and methanol 800:135 (v/v)

ample: eigh equivalent to average weight of tablet in 100 ml vol. flaskdd 60 ml diluent. Sonicate for 20 min. Dilute with same solvent &

Filter through 0.45 m filter paper.


50

100

150

200

250300

350

Intensity(mV) F

oli

cAcid

0

0 2 4 6 8 10




1 Folic acid 3.9 1.2 5713


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Purospher STAR LP Hydrochlorothiazide & Ramipril tablet



Injection: 0 L


Cell: 10 L


Mobile Phase (v/v): 5.6 g of sodium perchlorate in 500 ml water. pH adjust to 2.5 with ortho-phosphoric acid

0

50

100

150

200

250

300

350

Intensity(m

V)

Hydrochlorothiazide

Ramipril

emperature: 35 CDiluent: Mobile phase

tandard: eigh 10 mg of Hydrochlorothiazide & Ramipril separately and dilute to 100 ml

ith Acetonitrile. 5 ml of each solution mixed & dilute to 25 ml with mobile phase.


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1 Hydrochlorothiazide 2.8 1.1 3407

2 Ramipril 10.5 1.0 8524


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Purospher STAR LP Ivermectin Injection



Injection: 0 L


Cell: 10 L


Mobile Phase (v/v): Mixture of Acetonitrile:Methanol:Water 106:55:39 (v/v/v)

Temperature: mbient

Diluent: Methanol

Sample: 0.4 mg/ml in diluent


0

20

40

60

80

100

Intensity(mV)

H1B1b

H1B1a

0 10 20 30 40 50



No. Compound Time (min) Tailing Factor Resolution

1 H1B1b 28.0 1.1

2 H1B1a 38.0 1.1 7.9


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Purospher STAR LP Levofloxacin Tablet



Injection: 10 L


Cell: 10 L


Mobile Phase (v/v): 0.05 M Citric acid : 1M Ammonium acetate: Acetonitrile 84:1:15 (v/v/v)

empera ure:

Diluent: aterSample: Crush 20 tablets. Take the weight of tablet equivalent to 100 mg Levofloxacin. Dissolve

In 100 ml 0.1N HCl. Dilute 5 ml to 50 ml with water. Filter through 0.45m filter.


0

100

200

300

400

500

600

700

Intensity(m

V)

0 5 10 15 20 25 30 35 40




1 Levofloxacin 12.4 1.4 9691


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Purospher STAR LP Mecobalamine Injection



Injection: 10 L


Cell: 10 L


Mobile Phase (v/v): .72 g of KH2PO4 & 3.76 g hexane sulfonate in 1 L water.

pH adjust to 3.5 with ortho-phosphoric acid. Mix Buffer & Acetonitrile 80:20 (v/v)

20

40

60

80

100

Intensity(mV)

Mecobalamine

BenzylAlcoho

l

Temperature: 0 C

Diluent: Mobile phaseStandard: 5 mg of mecobalamine in 100 ml water. 5 ml of above solution & 50 mg of

benzyl alcohol diluted to 50 ml with water.

Sample: 5 ml of the smaple diluted to 100 ml with water

Precautions: Sample preparation do be done in dark. Use amber coloured flask & vial.


0

0 10 20 30 40




1 Mecobalamine 11.7 1.1 9526

2 Benzyl Alcohol 17.0 1.1 17457


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Purospher STAR LP Metformin and Pioglitazone Tablet



Injection: 10 L


Cell: 10 L


Mobile Phase (v/v): A: 1.38 g NaH2PO4 in 1800 ml water. Add 3.484 g of Sodium pentane sulfonate

pH adjust to 3.0 with ortho-phosphoric acid & dilute to 2000 ml with water.

100

200

300

400

500

600

700

800

Intensity(mV)

Pioglitazone

Metformin

B: Acetonitrile

Gradient Programme: See table

Temperature: 30 C

Sample Cooler: 4 C

Diluent: Mobile phase A and B 60:40 (v/v)

Sample: 200 ppm each in diluent


Time(min) %A %B

0.0 90 10

8.0 90 10

12.0 60 40

20.0 60 40

21.0 90 10

30.0 90 10

0

0 5 10 15 20 25 30



No. Compound Time (min) Tailing Factor Retention Factor

1 Metformin 4.9 1.3

2 Pioglitazone 15.9 1.2 2.3


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Purospher STAR LP Nebivolol Tablet



Injection: 20 L


Cell: 10 L


Mobile Phase (v/v): 6.8 g Potassium di-hydrogen phosphate in 1 L water. Add 2 ml triethyl amine

5

10

15

20

25

30

35

Intensity(mV)

Nebivolol

Adjust the pH to 3.0 with ortho-phosphoric acid.

Mix buffer, Acetonitrile & Methanol 45:25:30 (v/v/v)

emperature: Ambient


ample: Powder 20 tablets & weigh equivalent to 5 mg Nebivolol in 100 ml volumetric flask.Add 70 ml diluent & sonicate for 15 min. Dilute to 100 ml with diluent.Filter through 0.45 m filter paper


0

0 5 10 15 20Retention Time (min)



1 Nebivolol 9.5 1.1 10574


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Purospher STAR LP Ofloxacin Tablet



Injection: 10 L


Cell: 10 L

Flow Rate: .0 mL/min

Mobile Phase (v/v): 7.2 g of KH2PO4 in 1 L water. pH adjust to 2.4 with ortho-phosphoric

100

200

300

400

500

600

700

Intensity(mV

)

Ofl

oxacin

.

Temperature: 30 CDiluent: Methanol

Standard: 5 ppm in methanol

Sample: Crush 20 tablets. Weigh 25 mg equivalent of ofloxacin. Disperse in 25 ml methanol.

Sonicate for 25 min. Dilute to 100 ml with methanol & filter through 0.45 m filter paper.



0 10 20 30 40

Retention time (min)



1 Ofloxacin 20.6 1.3 6747

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Purospher STAR LP Olanzapine Tablet



Injection: 10 L


Cell: 10 L


Mobile Phase (v/v): 3 g of ammonium dihydrogen phosphate in 900 ml water. Add 2 ml of triethyl amine

Dilute to 1L with water. Adjust the pH to 2.5 with ortho-phosphoric acid.

100

200

300

400

500

600700

800

900

Intensity(mV) O

lanzapine

Mix Buffer & Methanol 70:30 (v/v)

emperature: 30 C

Diluent: 0.01 M HCl

tandard: 10 mg olanzapine in 25 ml acetonitrile. Dilute to 100 ml with 0.01 M HCl

ample: Crush 10 tablets. Weigh 10 mg equivalent of olanzapine. Disperse in 25 ml acetonitrile.

Sonicate for 25 min. Dilute to 100 ml with 0.01M HCl & filter through 0.45 m filter paper.


0

0 3 6 9 12 15




1 Olanzapine 4.9 1.2 3842


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Purospher STAR LP Hydrochlorothiazide and Olmesartan Tablet



Injection: 0 L


Cell: 10 L


Mobile Phase (v/v): 3.4 g of KH2PO4 in 1 L water. Sonicate & filter through N-66 Nylon membrane filter paper

100

200

300

400

500

600

700

Intensity(mV)

Hydrochlorothiazide

Olmesartan

emperature: 30 CDiluent: Buffer:Acetonitrile 50:50 (v/v)

tandard: 6 mg Hydrochlorothiazide & 80 mg Olmesartan diluted to 100 ml with diluent.

ample: eigh equivalent to average weight of the tablet. Dilute to 50 ml with diluent.

Sonicate for 30 min. Dilute 5 ml to 50 ml with diluent & filter through 0.45 m filter paper.


0

0 5 10 15 20




1 Hydrochlorothiazide 3.4 1.2 8061

2 Olmesartan 7.2 1.0 7238


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Purospher STAR LP Olmesartan and Amlodipine Tablet



Injection: 20 L


Cell: 10 L


Mobile Phase (v/v): 7 ml of tri-ethyl amine diluted to 1 L with water. pH adjusted to 3.0 with ortho-phosphoric acid

Mix Buffer & Acetonitrile 60:40 (v/v)

20

40

60

80

100

Intensity(mV)

Amlod

ipine

Olme

sartan

emperature: 40 C


tandard: 35 mg of amlodipine in 50 ml mobile phase. 20 mg of Olmesartan in 100 ml

volumetric flask. Add 10 ml of the above solution & dilute to 100 ml with mobile phase

ample: Weigh equivalent to average weight of the tablet in 100 ml volumetric flask.Add 70 ml of mobile phase & sonicate for 30 min. Dilute to 100 ml with mobile phase.Filter through 0.45 m filter paper.


0

0 5 10 15 20




1 Amlodipine 5.7 1.2 10408

2 Olmesartan 13.3 1.1 12345


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Purospher STAR LP Olmesartan Medoxemil Tablet



Injection: 0 LDetection: Shimadzu 2010 CHT, UV 225 nm

Cell: 10 L


Mobile Phase (v/v): 3.4 g of KH2PO4 in 1 L water. Sonicate & filter through N-66

Nylon membrane filter paper. Mix Buffer and Acetonitrile 55:45 (v/v)

0

50

100

150

200

250

Intensity(m

V)

Olm

esartan

Temperature: 30 C

Diluent: Buffer:Acetonitrile 50:50 (v/v)Sample: Crush 20 tablets & weigh equivalent to 20 mg Olmesartan in 100 ml

olumetric flask. Add 70 ml diluent & sonicate for 15 min.

Dilute to 100 ml with diluent. Further 5 ml dilute to 25 ml with the diluent.


0 3 6 9 12 15




1 Olmesartan 7.2 1.0 11844

Merck Millipore is a division of Merck KGaA Darmstadt Germany

purospher star lp_application guide

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