Analytical method development in HPLC guide

 

Analytical method development in HPLC

Written by 

Srinivas Birudukota 

This post is a guide and strategy on how to develop an analytical method in HPLC. It will instruct you on the design of the method, including developing a protocol for sample preparation and analysis. Finally, it will discuss the limitations of HPLC that must be taken into account when developing an analytical method. How does HPLC work? The principle of chromatography uses a moving phase (molecule) to separate two or more stationary phases (molecules). In this case, the stationary phase is a column filled with material that consists of positively charged particles that bind to and immobilize the analyte. The analyte is carried by a mobile phase which makes contact with both stationary phases. The mobile phase can flow back and forth between the two phases based on a specific gradient.

How to Convert specific optical rotation value to Enantiomer purity Value

How to Convert specific optical rotation value to Enantiomer purity Value

In Pharmaceutical compound Enantiomer purity is so much important which can  be only identified by Chiral HPLC chromatography (normal phase H!PLC)
the enantiomer or isomers (R-Isomer or S-Isomer) out of two isomers depends on the API only one should be desired and other is undesired.
If we have SOR value of the compound then we can easily calculate the isomer content
Below is the calculation

step-1

SOR/Purity of standard x 100

Step-2

100- step-1 result

Step-3

Step-2 result /2

step-4

S-isomer =step 1 + step 3

R isomer = 100- S isomer

CHROMATOGRAPHY AND ITS REVOLUTION

Chromatography become popular in Analytical Technique, most widely used by pharma industries and other sectors, the usage of chromatography is increasing day by day and spreded to other sectors of chemistry like oil, fuel,Polymer/Plastic, biological,Environmental etc.
Lets discuss about basic types Chromatography
1)TLC - Qualitative & semiquantitative Analysis,
2)HPLC-Qualitative & Quantitative Analysis,
3)GC-Qualitative & Quantitative Analysis,
And their are limitation in above mention techniques,
Modern Chromatography
1) HPTLC
2) LC-MS,UPLC
3) GC-HS,GC-MS,

HAEMOGLOBIN DETERMINATION

Procedure for the determination of Haemoglobin

Hemoglobin concentration can be measured in venous or capillary blood by colorimetric determination of derivatives of hemoglobin such as cyanmethemoglobin, oxyhemoglobin, or acid hematin. Various automated methods exist that are based on some of these principles. The preferred method, as recommended by the International Committee of Standardization in Hematology (14), involves the conversion of ail hemoglobin derivatives except sulfhemoglobin to cyanmethemoglobin by dilution of blood in a solution containing potassium cyanide and potassium ferricyanide. Absorbance is then measured in a photoelectric colorimeter or spectrophotometer at a wavelength of 540 nm. Reference standards of cyanmethemoglobin that conform to the specifications of the ICSH are commercially available.

Comparable information to that obtained from hemoglobin determinations can be obtained by the measurement of the packed red-cell volume, a simple technique that only requires a micro centrifuge and capillary tubes. However, due to changes in the mean corpuscular hemoglobin concentration that occur in iron deficiency anemia, changes in hemoglobin concentration are more marked than those in the packed cell volume.

Spectrophotometric Estimation of Aluminium at 389nm

Estimation of Aluminium

Introduction:

Provided a suitable pH is employed, a large number of colorimetric agents, among which aluminon may be particularly distinguished, combine with Al (III) to give coloration which are due not only to definite compounds but often also to adsorption compounds (lakes). The corresponding colorimetric determinations are not very precise since the results vary with the time, the grain size, etc. The pH is a very important factor. Many other substances, in particular Fe (III), give analogous coloration and in view of this, they should be separated out or complexed.

Oxinate:

Aluminium oxinate is yellow when dissolved in chloroform, carbon tetrachloride or benzene. The coloration is not very appreciable to the eye, but the adsorption is high in violet and ultraviolet light.

Sensitivity:

Ε=6,700 at 390nm and 80,000 at 260nm

Interfering ions:

Separation by extraction of the oxinate:

Aluminium oxinate can be extracted quantitatively at pH 4.5-5.0 (acetic buffer). In this way, aluminium may be separated from a number of elements, including Be(II), Th(IV) etc.

More specific separation may be obtained by operating in an ammoniacal medium at pH

9 in the presence of tartrate , cyanide and hydrogen peroxide. Separation from the following may thus be achieved : Cu(II), Co(II), Ni(II), Zn(II), Cd(II), Fe(III), Ti(IV), V(V), V(IV), U(VI), Mn(II), Cr(III), Mo(VI), Sn(IV), and Ag(I). Less than 10mg of Zr(IV) or  Nb (V) does not interfere.

Reagents:

Oxine, 2g in100ml of chloroform

Potassium cyanide, 13% in water

Sodium sulphite, 20% in water

Tartaric acid, 10% inn water

Hydrogen peroxide, 10volume

Standard solution of Al(III): 1g of 99% aluminium dissolved in hydrochloric acid.

Operating procedure:

2 ml of tartaric acid and 1ml of hydrogen peroxide are added to 25ml of a weekly acidic solution 10-150µg of Al(III). The solution is allowed to stand for 5min, and 5ml of sulphite are then added. After standing for a further 3 minutes, 10ml of cyanide are added and the solution is heated about 70°C-80°C and then cooled to 25-30°C. Finally, 2g of ammonium nitrate are added, and pH is adjusted to 8.9 ±0.3 with ammonia or Hcl. The solution is then transferred to a separating funnel, 5ml of oxine are added  ,and the funnel is shaken for 2min. The solution is allowed to settle, and the organic phase is collected in a measuring flask. The extraction is repeated three times, the solution being made upto 50ml with chloroform.

Finally , a colorimetric estimation is performed at 389nm in comparison with a blank.

Spectrophotometric Estimation of Bismuth 465nm or at UV region 337nm

Estimation of Bismuth

Introduction:

Iodide Complxes:

The iodine Complxes   of Bismuth (III) are orange colored, and Beers law is obeyed in the presence of an excess of I- ions (concentration of potassium iodide greater than   1%)

The complex is soluble in alcohols, esters, and ketones.

Sensitivity:

The molar coefficient ε ~ 34,000 at 337 nm in water

The coloration is stable for three to four hrs. The acidity should be fixed between the limit 1-2 N H2SO4

Interfering ions:

Oxidizing agents liberate iodine and should be reduced with e.g.: Sulpurous acid. CuI and AgI can be separated by precipitation without Loss of Bi (III), but PbI2 retains Bi (3) and interferences. Large amounts of   Cd (II) consume (2) I- by the formation of complexes .Hg (II) does so to an even greater extent .1000ppm of iron, 100 ppm of pb (II), 20 ppm of Cu (II) and 400 ppm of As, F-. and tartarate ions do not interfere

Pt (IV), Pd (II), Sn (IV) and Sb (III) produce interfering colors, but Sb (III) only interferes above 200 ppm, and the same doubtless applies to Sn (IV) .Sb (III) and Bi (III) can be estimated simultaneously, Cl- and F- weaken the coloration

REAGENTS:

Potassium iodide, 10% in water;

Sulphurous Acid solution, 5% freshly prepared;

Hypo phosphorous acid, 30% in water

Operating Procedure:

The initial solution consists of 10-20ml, containing from 5-50μg of bismuth (III). The acidity should be adjusted to 1-2N H2SO4. Then .0.1ml

Of sulphurous acid, 1ml of hypo phosphorous acid, and 3ml of iodide are added and the volume made up to 25ml. Colorimetry is performed at 465nm, or alternatively in the UV at 337nm

The blank test or the calibration curve should be determined under identical conditions with respect to acidity and the concentration of salts and iodine 

Spectrophotometric Estimation of Molybdenum at 475nm

ESTIMATION OF MOLYBDENUM

Complex thiocyanate  of Mo(V)

The controlled reduction of Mo(VI) in the presence of thiocyanate ions leads to the formation of a complex orange –red Mo(V) thiocyanate. The reduction should  not be too vigorous and the acidity should be fixed at a definite level, since several alternative reductions may occur. Thus for example , Mo(III) may be formed or, in a weakly acidic solution ‘molybdenum blue’ may appear.

Sensitivity:

Interfering Ions:

Colored ions may be interfere if the molybdenum is not first isolated by extraction . large amounts of Cr(III) should be separated in the form of CrO₂Cl₂

Certain other ions give rise to extractable colored thiocyanate complexes; moderate quantities of Fe(III) are reduced and to do interfere. Pt(IV) interferes. Co(II) interfere if its content exceeds half that of the Mo(IV). Cu(II precipitates in the form of cuprous thiocyanate and may be separated in this way. W(IV) should be complexed by citrate or tartrate ions, and Ti(IV) by F ^–

Large amounts of Bi(III), V(V) and P(V) interfere. Re(VII) gives the same reaction. U(VI) interferes

Reagents:

Potassium thiocyanate, 10%

Stannous chloride: 10g of SnCL₂. 2 H₂O are dissolved in 10ml of concentrated HCL and the solution is made up to 100ml with water.

Solutions of ferrous ion:  1 g of Mohr’s salt is dissolved in 100ml of 0.2N( I/80) sulphuric acid.

Isoamyl alcohol.

Standard solution of molybdenum: 750 mg of guaranteed purity MoO₃ are dissolved in a few ml of dilute caustic soda. The solution is made slightly acid with HCl and the volume adjusted to 500ml. The solution id diluted to the point where 1 ml contains 1 µg of Mo(VI).

Operating Procedure:

2.0 ml of concentrated HCL . 1ml of ferrous solution, 3.ml of thiocyanate, and 3ml of stannous chloride are added to 15ml of solution containing from 1 to50µg of molybdenum. The solution is diluted to 25ml and an accurately measured 10ml portion of iso-amyl alcohol is added. After shaking vigorously for 1 minute and allowing to settle , the colorimetry is performed at 475nm