Investigation of the precision and accuracy of various analytical techniques in determining the identity and quantity of barbiturates in a mixture.

Aim

The aim of this investigation was to identify the barbiturates given to us in a mixture (Mixture A) and to quantify the amounts present using a variety of analytical procedures for confirmation of results. Some techniques were used for preliminary investigation.

Qualitative analysis

Thin layer chromatography (TLC), infra red (IR) and Raman spectroscopy were used in determining which barbiturates were present in the mixture.

A melting point determination was not done however because the melting point of a mixture of compounds will be less than the melting point of the individual compounds so therefore

TLC analysis

An analyte migrates up a layer of stationary phase under the influence of a mobile phase, which moves through the stationary phase by capillary action. The distance moved by the analyte is determined by its relative affinity for the stationary vs. the mobile phase.

The sample was spotted onto a silica plate and was run alongside samples of known barbiturates in 3 different mobile phases

Dichloromethane: acetone (9:1)
Chloroform: acetone: ammonia (9:9:2)
Propan-2-ol: chloroform: ammonia (9:9:2)

The plates were exposed to ammonia vapour before using fluorescence quenching as a detection method.

From the plates it can be seen that phenbarbitone (PB) and barbitone are the barbiturates present in mixture A. See fig 1 (write on plates how you came to conclusion)

IR analysis

Mixture A and standards of different barbiturates were analysed by the IR spectrophotometer by radiation at wavelengths ranging from 500cm-1 to 4000cm-1.

The IR spectra of Mixture A shows a 95.53% match with phenobarbitone and otherwise appears not to contain any other barbiturates.

This could be because there is much more phenobarbitone than barbitone in the mixture but looking at the TLC plates this does appear to be the case. The spots of barbitone and phenobarbitone are similar in size.

Another reason may be that the machine had not been properly cleaned before use. This is unlikely because Mixture A was analysed twice and both spectra produced a similar result. See fig 2

To be sure the spectrum of a standard could be subtracted and the remainder identified.

Raman analysis

The Raman effect is analogous to fluorescence except that it is not wavelength dependant and does not require the molecule to have a chromophore. The energy shift in cm-1 due to inelastic scattering of laser radiation is measured rather than wavelength. The shifts measured correspond to the wavenumbers of the bands present in the middle-IR spectrum of the molecule.

Mixture A and standards of phenobarbitone and barbitone were put through the Raman spectrometer.

The spectrum produced of Mixture A shows a 77.43% match with phenobarbitone and a 54.74% match with barbitone standards on the machine’s library.

When the barbitone spectrum was subtracted from the Mixture A spectrum, a 99.28% correlation with phenobarbitone is obtained.

When the phenobarbitone spectra was subtracted from the Mixture A spectrum, a 98.64% correlation with barbitone was obtained.

Therefore the result of the Raman analysis suggests that phenobarbitone and barbitone are the barbiturates present in the mixture.

Quantitation methods

Gas liquid chromatography (GLC) analysis, high-pressure liquid chromatography (HPLC) and ultra-violet (UV) spectroscopy were used in order to quantify the amount of phenobarbitone and barbitone present in mixture A

GLC analysis

A gaseous mobile phase flows under pressure through a heated tube either coated with a ...

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When the phenobarbitone spectra was subtracted from the Mixture A spectrum, a 98.64% correlation with barbitone was obtained.

Therefore the result of the Raman analysis suggests that phenobarbitone and barbitone are the barbiturates present in the mixture.

Quantitation methods

GLC analysis

A gaseous mobile phase flows under pressure through a heated tube either coated with a liquid stationary phase or packed with a liquid stationary phase coated onto a solid support. The analyte is loaded onto the head of the column via a heated injection port where it evaporates. It then condenses at the head of the column, which is at a lower temperature. The oven temperature is then either held constant or is programmed to rise gradually. Once on the column separation of a mixture occurs according to the relative lengths of time spent by its components in the stationary phase. Monitoring of the column effluent can be carried out with a variety of detectors.

An internal standard was used in the investigation to eliminate the errors created by variations of injector volume. The internal standard should have a similar structure to the unknown, must produce a distant peak and t must not be present in the sample. In the investigation butabarbitone was used.

An initial run was done to determine the retention times of the compounds

Leave space for photocopy

Because the internal standard was not used with the unknown, peak area instead of ratio was plotted against concentration.

The R2 value obtained for the barbitone calibration plot was 0.2597, which is very poor. The R2 value obtained for the phenobarbitone was 0.7298, which is not very good either.

From the calibration plots, the concentration of barbitone is (16.924-8.09440)/615.62 = 0.0143 % w/v and the concentration of phenobarbitone is 24.2709/5889.9 = 0.00412% w/v.

HPLC analysis

A liquid mobile phase is pumped under pressure through a stainless steel column containing particles of a stationary phase with a diameter of 3-10um. The analyte is loaded onto the head of the column via a loop valve and separation of the mixture occurs according to the relative amount of time spent by its components in the stationary phase. It should be noted that all components in a mixture spend more or less the same time in the mobile phase in order to exit the column. Monitoring of the column effluent can be carried out with a variety of detectors.

The mobile phase used in the investigation was a 60% methanol, 40% water mix. Phenobarbitone and barbitone were dissolved into solutions of varying concentrations in a 1:1 ratio. The methanol/water mix was used to make up the solutions at concentrations of 1, 2, 3, 4, 5 and 6 mg/100ml.

The first run was done to determine the

A calibration plot for the two barbiturates were constructed from the data collected at each concentration.

A good correlation was obtained for both calibration plots. R2 value for barbitone was 0.9977 and the R2 for phenobarbotone was 0.9993.

The amount of barbitone in the mixture a solution is;

604316 = 213463x + 57408

546908 = 213463

x = 2.5620

2.56 mg/100ml

The amount of phenobarbitone in the Mixture A solution is

754013 = 288986x + 14325

739688 = 288986x

x = 2.5596

2.56mg/100ml

From these results, it can be concluded that phenobarbitone and barbitone are present in Mixture A in equal amounts, i.e. a 50:50 mix.

Table

UV analysis

Radiation in the wavelength range 200-700 nm is passed through a solution of a compound. The electrons in the bond within the molecule become excited so that they occupy a higher quantum state and in the process absorb some the energy passing through the solution. The more loosely held the electrons are within the bond of the molecule the longer the wavelength of the radiation absorbed.

UV detection is used in combination with HPLC to provide an accurate, precise and robust method for quantitative analysis of pharmaceutical products and is the industry standard method for this purpose.

This method didn’t work well when dissolved in methanol, the spectra obtained were very poor. When NaOH was added, better results were achieved. Because the spectra of both compounds shifted and were too similar, difference spectroscopy could not be done.

The concentration of phenobarbitone was calculated using

Csample x (peak- trough)standard = Csample x (peak –trough)sample

Csample = 0.005 x (0.013791 - -0.013781)/ (0.06110 - -0.053968)

= 1.2mg/100ml

Conclusion

The best technique used in determining which compounds were present in mixture A was Raman spectroscopy.

HPLC was the most accurate and precise technique used in the quantitation of the amounts of barbiturates present. It was also the most sensitive and selective technique employed.

Discussion

The first part of the investigation was done to determine which barbiturates were present in mixture A. The techniques employed were TLC, IR and Raman spectroscopy.

TLC

TLC analysis determined that phenobarbotine and barbitone were present in mixture a. the advantages of using TLC include

Detection by a chemical reaction with a visualisation reagent can be carried out, which means that almost every type of compound can be detected if a suitable detection reagent is used.
Robust and cheap.
Can be used as a quantitation technique for compounds with no chromophore
Since all the components in the chromatographic system can be seen, there is no risk, as in the case in GLC and HPLC analyses, that some components are not observed because they do not elute from the chromatographic system.
Batch chromatography can be used to analyse many samples at once, increasing the speed of analysis, and can be automated.
The method is flexible since the TLC plates can be simply treated with a variety of chemicals thus imparting a wide range of properties to the stationary phase.

However TLC has its limitations

The number of theoretical plates available for separation is limited in routine TLC systems.
Sensitivity often limited
Requires more operator skill for optimal use than TLC
Not suitable fro volatile compounds

IR result suggested that only phenobarbitone was present in mixture A and further investigation proved this not to be case. IR did not appear to very useful but it does have its strengths

It provides a complex fingerprint which is unique to the compound being analysed
Computer control of instruments means that matching of the spectrum of a compound to its standard fingerprint can now be readily carried out.

IR has some limitations

It is rarely used as a quantitative technique because of relative difficulty in sample preparation and the complexity of the spectra.
Usually, it can only detect gross impurities in the samples
The technique is lacking in robustness since sample handling can have an effect on the spectrum obtained and thus care has to be taken in sample processing.

Raman

The spectra obtained from Raman analysis were good. They clearly show that phenobarbitone and barbitone are the compounds present in mixture A.

Raman spectroscopy is complementary to middle-IR spectroscopy but requires very little sample preparation since near infra-red with its good penetration properties can be used for the analysis.

Unfortunately, Raman spectroscopy is not yet fully established as a quantitative technique and solvents used may interfere when samples are run in solution.

In the next stage of the investigation, GLC, HPLC and UV analysis was done to determine the amount of barbiturates in Mixture A.

GLC

GLC is in pharmaceutical analysis in the characterisation of some unformulated drugs, particularly with regard to detection of process impurities. GLC is used when conducting limit tests for solvent residues and volatile impurities in drug substances. It is also used in characterisation of raw materials used in synthesis of drug molecules.

Advantages of using GLC

It is capable of the same quantitative accuracy and precision as HPLC, particularly when used in conjunction with an internal standard
Has much greater separating power than HPLC when used with capillary columns
Can be used to determine compounds with no chromophore
The mobile phase does not vary and does not require disposal and, even if helium is used ad the carrier gas, is cheap compared to the organic solvents used in HPLC

GLC has its limitations

Only thermally stable and volatile compounds can be analysed.
The sample may require derivatisation to convert it to a volatile form, thus introducing an extra step in analysis and potential contaminants.
Quantitative sample introduction is more difficult because of the small sample volume injected.
Aqueous solutions and salts cannot be injected into the instrument

During this investigation, the results obtained from GLC analysis were not good. The calibration plots constructed had poor R2 which suggests that the method was not accurate or precise. There are several reasons why this could have happened. GLC analysis requires more operator skill than techniques such as TLC and mistakes could have been made here. There could have been contamination of the sample due to residues left in the injector. From the results of this investigation, I would not recommend the use of GLC for the analysis of barbiturates or similar compounds.

HPLC

HPLC is the technique most commonly used for the quantitation of drugs in formulations. HPLC has several strengths

Easily controlled and precise sample introduction ensures quantitative precision.
HPLC is the chromatographic technique, which has seen the most intensive development in recent years leading to improved columns, detectors and software control.
The variety of columns and detectors means that the selectivity of the method can be readily adjusted.
Compared to GLC there is less risk of sample degradation because heating is not required in the chromatographic process.
HPLC is readily automated.

HPLC has some disadvantages

There is still a requirement for reliable and inexpensive detectors which can monitor compounds that lack a chromophore.
Drugs have to be extracted from their formulation prior to analysis.
Large amounts of organic solvent waste are generated, which is expensive to dispose of.

Straight lines were achieved in the calibration plots and the R2 values were0.9993 and 0.9977, which are both very good. The intercepts were relatively small.

The result shows that HPLC is a precise and accurate technique in the quantitation of drugs in formulation.

UV analysis has several applications in pharmaceutical analysis. It is used in the determination of the pka, partition coefficients, solubility and the release of drugs from formulations with time.

The UV spectrum of a compound is often used as one of the number of pharmacopoeial identity checks.

Strengths

An easy to use, cheap and robust method offering good precision for making quantitative measurements of drugs in formulations
Routine method for determining some of the physico-chemical properties of drugs which need to be known for the purposes of formulation.
Some of the problems of the basic method can be solved by the use of derivative spectra.

UV limitations

Only moderately selective. The selectivity of the method depends on the chromophore of the individual drugs, e.g. a coloured drug with an extended chromophore is more distinctive than a drug with a simple benzene chromophore.
Not readily applicable to the analysis of mixtures.

Derivative spectroscopy was used because there was a lot of background interference. There was still some interference even at the 4th derivative (D4). Spectra were repeated several times but with similar results. This led me to believe that the machine was faulty and another machine were used the results may have been different.