Category Archives: Pharmaceutical Analysis – I (Theory)

PRIMARY AND SECONDARY STANDARDS

PRIMARY AND SECONDARY STANDARD

INTRODUCTION

In life, we always hear and use the word ‘standard’ e.g. the standard of rail service or the standard of education. What is the meaning of this word? Here we will discuss the standard, its types and properties and its uses.

STANDARDS

In pharmaceutical analysis, the word standard means a material which contains a substance of our interest with a known concentration. By using definite numbers with proper units we can express this. By using this standard we can determine the concentration of that substance in a new material. Therefore, primary standard is used as primary calibrator or primary reference material.

  1. Functions

Therefore, standard has the following uses in pharmaceutical analysis laboratory-

(a)   The standard is used as a reference by which we can determine unknown concentration

(b)  Used for standardization of  volumetric solutions

(c)  Used in the Preparation of secondary standard

(d)  Used to calibrate an instrument.

  1. Types

Standards can be divided into two types:

  1. Primary standard
  2. Secondary standard

Primary standard

A primary standard is a chemical or reagent which has certain properties such as-

(a)   It is extremely pure – A primary standard material should be extremely pure which means that it should be a chemical of high grade of purity, preferably 99.98%. In a pahramceutical analysis laboratory we  come across chemicals of different grade of purity. If we check the label we will notice a number with percentage termed as purity. So when a chemical has purity of 99.98% or more it is a suitable material to be used as  primary standard.

(b)  It is highly stable – It should be highly stable which means it usually does not react easily when kept in its pure form or it should have very low reactivity. This is important because if a reagent reacts easily with atmospheric oxygen or water or changes its property over time then it is unreliable and such a unstable and unreliable chemicals can never be used as standard.

(c)   It is anhydrous- It should be anhydrous which means that it does not contain any water molecule in its molecular structure. For example, in a pharmaceutical analysis laboratory we come across same chemical with different number of water molecules attached with it e.g. magnesium sulphate (MgSO4), which is also called Epsom salt. The Epsom salt which is found in drug store is a chemical with formula MgSO4.7H2O. Therefore if we want to prepare a primary standard of magnesium sulphate we should purchase an anhydrous MgSO4 preferably an analytical reagent grade chemical and with purity greater than 99.98%.

(d)  It is less hygroscopic  in nature-  The chemical preferably should be less hygroscopic  i.e. on opening the container it should not absorb water molecules from atmosphere.

(e)   It has very high molecular weight- It has very high molecular weight compared to its other similar forms. For example Epsom salt. Take 1 gram of MgSO4 for making a primary standard and name it as salt A. Now take 1 gram of MgSO4.7H2O for common uses and name it as salt B. Now if we compare the actual weight (Molecular weight) of magnesium sulphate to make a standard solution for both chemicals then it is found that-

CHEMICALS MOLECULAR WEIGHT
MgSO4 108
MgSO4.7H2O 234

 

In first case, molecule of salt A the weight of actual MgSO4 will be 108 atomic mass unit. But in second case, molecule of salt B the weight of actual MgSO4 will be 108 out of its total weight of 234 atomic mass unit.

108 gram salt A (MgSO4) will give 108 gram of MgSO4

So, 1 gram MgSO4 salt will give = 108/108 = 1 gram of MgSO4

But 234 gram salt B (MgSO4.7H2O) will give 108 gram of MgSO4

So, 1 gram MgSO4.7H2O salt will give = 108/234 = 0.461 gram of MgSO4

– Therefore if by mistake we make a standard out of salt B, actually we are taking 0.461 gram of MgSO4 and calculating it as 1 gram. So with this faulty standard estimation of MgSO4 in other unknown solution will give less result than the actual concentration. Hence it is important that primary standards must be anhydrous and of high molecular weight.

(f)   It can be weighed easily – It can be weighed easily because it is so pure that its weight is in fact a true representative of number of moles present in its actual weight.

(g)  It should be ready to use and available

(h)  It should be preferably non toxic

(i)   It should not be expensive

Uses – Primary standard is used to standardize a volumetric solution i.e. they are used for standardization of titration of solutions. It can be used for titration of acids as well as bases. In a pharmaceutical analysis laboratory, for acid titration the most common basic chemical standard is sodium carbonate (Na2CO3), (TRIS) Trisaminomethane [(CH2OH)3CNH2] etc. For base titration, potassium hydrogen phthalate [(KHP): KHC8H4O4] etc. For redox titration, potassium dichromate (K2Cr2O7) & Sodium oxalate (Na2C2O4) are very often used as primary standard.

– The primary standard is used for calibration of secondary standard or for method validation using a  specific method.

Secondary standard

A secondary standard is involved in preparation of reagents and kits or laboratories responsible for producing quality control material for other laboratories. They use primary standard as the primary calibrator or primary reference material. Secondary standard is used for the purpose of calibration of control material in laboratory for analysis of unknown concentration of a substance. So basically, secondary standard serves the purpose of external quality control for laboratories. So it is essential that the secondary standard must first be standardized against the primary standard.

For preparation of secondary standard solution, aqueous solution must be of high grade purity. It must be deionized, if water is used as aqueous solvent.

A secondary standard is a chemical or reagent which has certain properties such as

(a)   The purity of secondary standard is less than primary standard

(b)   Secondary standard  is less stable and more reactive than primary standard

(c)   The secondary standard solution remains stable for a long time

(d)   Secondary standard is titrated against primary standard

Example – 1. Anhydrous sodium hydroxide (NaOH). It is extremely hygroscopic. As  soon  as  the  bottle  is  opened,  NaOH  absorbs moisture from atmosphere and it becomes moist. Lets do it practically, take an analytical balance and place a Petridish and make its weight as zero (Tare). Now open the NaOH bottle and place little NaOH crystal on the petridish and note the weight. Now keep the glass windows of the analytical balance open for few minutes and notice the gradual increase in its weight. This is because the NaOH crystals absorb water molecule from air.

  1. Potassium permanganate (KMnO4) very often used as secondary standard. It is a good oxidizing agent, that’s why reactive and hence less stable. Its own oxidized product manganese oxide (MnO2) contaminates the content. Hence it is unsuitable for being a primary standard.

–  Secondary standard is used as a calibrator by smaller laboratories involved in actual analysis of unknown samples.

Calibration – Calibration means to check whether an instrument is working properly or not i.e. the instrument is giving correct measurement or not. Calibration and standardization are synonyms of each other but in case of solution we use the word standardization and in case of instruments  we use the calibration . This is the process by which we compare the measurements by a standard or an instrument (primary) with another standard or an instrument (secondary). By doing so, we try to eliminate any variation or difference in measurement by the secondary standard or an instrument.

–  Calibration using titration:

Example – Estimation of vitamin C in lemon. For this estimation, ascorbic acid is used as standard. For estimation first of all, take ascorbic acid and prepare a standard solution of known concentration (for example 40mg/dl). Now prepare a diluted solution of indicator called 2 Di -chloro indophenol (2- DCIP) and add ascorbic acid drop by drop till it is completely decolourized e.g. 20 ml.

100 ml ascorbic acid standard solution has 40 mg of ascorbic acid in it

1 ml of standard solution has = 40/100 = 0.4 mg ascorbic acid

Now 20 ml of standard solution has = 0.4 ×20 = 8 mg ascorbic acid

This says that our 2- DCIP requires 8 mg of pure ascorbic acid for its complete neutralization or titration.

Next let us use our lemon. Weigh the lemon. Let us say it weighs 4 g. add lemon juice drop by drop. Let us say it took several drops of lemon juice for complete neutralization of 2- DCIP. Now weigh lemon and now it weighs 2 g. So it proves that 2g of lemon juice present in 4 g of lemon has 8 mg of ascorbic acid. Why 8 mg of ascorbic acid? Simple. This is so because; 8 mg of ascorbic acid is required to completely neutralization 2 DCIP. So from this we came to know that 4 g of lemon has 8 mg of ascorbic acid

So 100 g of lemon would contain = 50 mg of ascorbic acid

So concentration of ascorbic acid in our variety of lemon is 50 mg/100 g that is equal to 50 mg%.

–  The secondary standard is used for calibration of control materials in a lab using a reference method and it is 0closer to the true value of the substance. So it is used for external quality control programme.

–  Whereas in a clinical laboratory unknown samples are analyzed against control materials using a routine/ field method

POINTS TO REMEMBER

  • There are two types of standard.
  • A primary standard should be >99.8% pure.
  • Secondary standard is more reactive, on comparing primary and secondary standards.
  • Secondary standard or an instrument can be calibrated using primary standard.
  • By titration using primary standard, secondary standard can be prepared.
  • A standard also called a calibrator is a material containing a known substance with known concentration.
  • Standard is of two types primary standard and secondary standard.
  • If we compare primary and secondary standard, then it is found that Primary standard is purer, more stable, less reactive, anhydrous and less hygroscopic compared to secondary standard.
  • Secondary standard is titrated or calibrated against primary standard and used as a reference material in laboratories for the purpose of analysis.
  • To get quality solution, pure and de-ionized aqueous solvent should be used to prepare standard solution.
  • In laboratory, primary standard acts as primary reference material.
  • Secondary standard is used as an external quality control for calibration of internal quality control in laboratories.

SIGNIFICANT FIGURES & RULES

SIGNIFICANT FIGURES

The significant figures of a number are digits that carry meaning contributing to its measurement resolution.

There are certain rules to find out the significant figures of a given number-

Rules 1 – All non-zero digits are significant.

Example –

  1. 91 – In 91, there are two significant figures 9 & 1.
  2. 123.45 – Here, there are five significant figures 1,2,3,4 & 5.

Rule 2 – Zeros between non-zero digits are significant.

Example- 101.1203 – As per the rule, there are seven significant figures in 101.1203, these are 1,0,1,,1,2,0 & 3.

Rule 3 – Leading zeros are never significant.

Example – 0.00052 – There are only two significant figures 5 & 2.

Rule 4 – In a number with a decimal point, trailing zero those to the right of the last non-zero digit are significant.

Example – 12.2300 – Here, there are six significant figures 1,2,2,3,0,& 0.

Rule 5 – In a number without a decimal point, trailing zeros may or may not be significant.

Example  – 1300 – In 1300, there are four significant figures 1,3,0 & 0.

 

PHARMACEUTICAL ANALYSIS, QUALITATIVE & QUANTITATIVE ANALYSIS, METHODS OF ANALYSIS

PHARMACEUTICAL ANALYSIS

Pharma means drug and analysis means determination or estimation. So pharmaceutical analysis means determination or estimation of drug in a simple way. But in the language of science, pharmaceutical analysis is defined as it is the branch of science which deals with the study of separation, identification and quantification of drugs (analyte). The person who is involved in analysis, is known as analyst.

Example – i. A mixture when analyzed gives compounds and when a compound is analyzed gives elements. Element is the basic unit of any substance.

  1. separation of protein into its individual components (amino acids), its identification and then quantification.

QUALITATIVE & QUANTITATIVE ANALYSIS

Qualitative Analysis – The word qualitative is made from quality. In qualitative analysis tests are performed on the sample to know its composition i.e. constituents present in the sample. Simply we can say qualitative analysis is the identification of the analyte.

Example – Suppose a sample (Z) is to be analyzed.

When Z is analyzed, the analyst found 5 constituents A, B, C, D & E in that sample. So determination of constituents only not its amount is called qualitative analysis.

Quantitative analysis – the word quantitative is made from quantity. In quantitative analysis tests are performed on the sample to know its composition along with its quantity/amount  i.e. in how much quantity, constituents are present in the sample. Simply we can say the quantitative analysis is determination of amount or concentration of analyte.

Methods of Analysis

  1. Chemical method – It is also called wet analysis, which is based on titrations i.e. reactions between titrant and titrand. Lot of titrations are their which have their own working principles e.g. acid ase titration, redox titration, precipitation titration, complexometric titration etc.
  2. Instrumental method – it includes spectroscopic and chromatographic methodsI. i. Spectroscopic method – in this method specific instruments are employed which works on the principle of absorption and emission of electromagnetic radiations g. Ultraviolet-Visible spectroscopy (UV/Vis), Infrared spectroscopy (IR), Nuclear magnetic resonance spectroscopy (NMR). ii. Chromatographic method – Here we use affinity or partition coefficient differences between drugs e.g. Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), Gas chromatography (GC).

ACCURACY, PRECISION & ERRORS -STATISTICAL TOOLS OF MEASUREMENT

STATISTICS INVOLVED IN PHARMACEUTICAL ANALYSIS

Statistics is of great importance in calculating the result from the analytical data obtained. These are accuracy, precision, errors and significant figures. To understand accuracy, precision and error some terms should be very clear in mind i.e. observed value & standard value.

Observed value – These are the values which an analyst obtains after analysis of sample e.g. an analyst after analysis of a sample says that the sample is 80% pure. This 80% is observed value.

Standard value – Sometimes it is also called as true value. This is the value which the sample claims e.g. a tablet sample claims paracetamol 100 mg. This 100 mg is standard value.

Accuracy, Precision & Errors

These can be easily explained by an example. Two analysts (Analyst 1 & analyst 2) are analyzing a sample five times using same method and instruments (sample claims it is 100% pure i.e. standard value is 100%). The results  of the of the tests performed by the two analysts are given in the table below-

TEST NUMBER ANALYST 1 ANALYST 2
1 99.10 % 98.95 %
2 99.30 % 98.91 %
3 99.80 % 98.96 %
4 99.50 % 98.92 %
5 100.00 % 98.93 %

Now, if we calculate the average/mean value of the results obtained by analyst 1, we get

99.10 + 99.30 + 99.80 + 99.50 + 100.00

Mean = —————————————————————— = 497.7 / 5 = 99.54 %

5

Error = 100 – 99.54 = 0.46 %

Now, if we calculate the average/mean value of the results obtained by analyst 2, we get

98.95 + 98.91 + 98.96 + 98.92 + 98.93

Mean = —————————————————————— = 494.67 / 5 = 98.934 %

5

Error = 100 – 98.934 = 1.066 %

Accuracy – Accuracy means how near the observed value is to the standard value.

Precision – Precision means nearness between several measurements of the same quantity.

If we compare the results of the two analysts, error of analyst 1 is 0.46% and of analyst 2 is 1.066%, following things can be concluded-

  1. Results of analyst 1 is more accurate than the results of analyst 2 because comparably, error of analyst 1 (0.46%)is much less than the error of analyst 2 (1.066%)
  2. Results of analyst 2 is much more précised than the results of analyst 1 because there is very less difference in each other.

Now we will discuss about error, its types and methods of minimizing error.

ERROR

Error – The difference between the observed value and the standard value. Larger the difference, more be the error. If two analysts performs a analysis using the same instrument and for measurement, its not essential that both will get the same results. There may be difference in their measurements. This difference is called as error.

Types of error

Broadly we can divide error into three types namely personal error, systematic error and random error.

  1. Personal error – it arises due to the use of faulty procedure. For example
  2. two persons if taking reading of thermometer, there readings may differ.
  3. two persons if pipetting some chemicals, quantity may differ because some use lower meniscus and some upper meniscus while pipetting.
  • during titration, if the end point is pink color, a person can do mistake because of the confusion in colors like light pink or dark pink.
  1. Systematic error or determinate error – it arises due to instrumental fault i.e. use of uncalibrated (calibration means to check the machine whether it working properly or not, if not it will be corrected) instruments.

Example – if a person is using a digital weighing machine to weigh a sample and the machine is giving weight 50 mg. he takes the weight of the sample as 50 mg. This reading may be wrong because may e the machine is uncalibrated, may be the correct result is 48 mg or 52 mg. This type of error comes under systematic error.

  1. Random error or indeterminate error – These errors are beyond our control and arises due to sudden change in environmental conditions like
  2. Sudden change in temperature, humidity
  3. Voltage fluctuation

Sources of error

Human error and equipment error are the sources of error.

  1. Human error – Examples of human error are-
  2. Incorrect measurement
  3. Use of contaminating glasswares e.g. a person uses a pipette to pipette 5 ml nitric acid and without washing he uses it for pipetting hydrochloric acid. Here the hydrochloric acid gets contaminated.
  • Dirty working place, glasswares, instruments
  1. Equipment error – taking measurement without checking, its giving correct reading or not.

Methods for minimizing errors –     

  1. Correct measurements
  2. Working place and instruments should be clean
  3. Glasswares should be washed, cleaned and dried.
  4. Calibrated instruments should be always used
  5. Use of correct procedure
  6. Running a blank determination
  7. Use of two methods and results are compared

PHARMACEUTICAL ANALYSIS

Pharma means drug and analysis means determination or estimation. So pharmaceutical analysis means determination or estimation of drug in a simple way. But in the language of science, pharmaceutical analysis is defined as it is the branch of science which deals with the study of separation, identification and quantification of drugs (analyte). The person who is involved in analysis, is known as analyst.