FYP

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Table of contents Page
Abstract…………………………………………………………………………….....2
1.0 Introduction……………………………………………………………………....4
1.1.0 UV Filters……………………………………………………………………….6
1.1.1 History of UV Filters…………………………………………………………....7
1.1.2 Regulation of UV Filters………………………………………………………..8
1.1.3 Mechanism of Action of UV Filters……………………………………………13
1.1.4 Developments in UV Filter Technology………………………………………..14
1.2 Scope of High Performance Liquid Chromatography……………………………16
1.2.0 Eluents in HPLC……………………………………………………………......19
1.2.1 Retention Time………………………………………………………………….21
1.2.2 HPLC Pumps…………………………………………………………………....22
1.2.3 Sample Injection...................................................................................................24
1.2.4 Columns…………………………………………………………………………26
1.2.5 HPLC Detectors………………………………………………………………....27
1.2.6 HPLC Modes……………………………………………………………………29
1.2.7 Quantitative Analysis…………………………………………………………....31
2.0 Literature review…………………………………………………………………..32
3.0 Batch Books……………………………………………………………………….42
4.0 Standard Operating procedures (SOPs)…………………………………………...55
5.0 Calculations………………………………………………………………………..77
6.0 Results and Discussion…………………………………………………………….116
7.0 Conclusion…………………………………………………………………………126
8.0 References…………………………………………………………………………129
9.0 Appendix…………………………………………………………………………..

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In this project cosmetic creams were manufactured. From here UV filters with a calculated
SPF (Sun Protection Factor) rating were added. These SPF factors were calculated through
the BASF website which provided an SPF simulator where one could enter the percentage
amounts of UV filter and in return receive an SPF factor for those particular filters. There
was six UV filters analysed by High Performance Liquid Chromatography (HPLC) using a
C18 BDS column with an isocratic mobile phase composition of 70% ethanol 30% 1% acetic
acid. The filters were added into the formulation and extracted back out in order to see if they
would be evenly dispersed about the formulation.
An extraction technique was employed to quantify the amount of UV filter present in the
sample. These filters were added to the formulation in various quantities which were
calculated from the SPF sunscreen simulator and were analysed to determine if the quantities
that were being added were evenly distributed about the cream. A quantitative analysis was
carried out to calculate the exact amount of the UV filters of interest contained in the sample
and this was compared to the amount the filter that was put into the formulation in the
beginning in order to determine the percentage recovery.
Three different formulations comprising of approximately 9% beeswax, 0.6% borax, 25% oil
(mineral/vegetable) and 20% water were decide upon. For formulation 1 an SPF factor of
17.7 was calculated. The three filters added were BDM 4%, MBC 3% and BZ3 6%. The
percentage recoveries obtained for these three filters were BDM 2.5%, 2% for MBC and
3.62% for BZ3. In the manufacture of formulation 2 an SPF factor of 15.2 was calculated.
The UV filters used in this formulation were OS 5%, OMC 5% and BDM 9%. The
percentage recoveries obtained for these three filters were for the BDM 3.87%, 1.86% for
OMC and 2.56% for OS. For the final formulation three UV filters were added namely BDM
4%, OCR 5% and MBC 3% and an SPF factor of 17.5 was calculated from these percentages.
The average per cent recovered of OCR, BDM and MBC in the samples was calculated to be
1.48%, 1.68% and 1.89% respectively.

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The aim of this project involved the manufacture of cosmetic creams and the subsequent
analysis and extraction of a number of UV filters most notably BDM, OMC, OS and BZ3
using certain HPLC parameters from a method developed by Mary Wharton et al. These
parameters included the column (C18 BDS), wavelength (313nm) and mobile phase (ethanol:
1% acetic acid). Also another aim in this project was to gain proficient use in the operation of
the HPLC instrument.
During honey production beeswax is produced which is a by-product of the honey
production. The applications of the beeswax include; hand lotions, lip balms, moisturizers,
hand creams, waxes and dental moulds. The female worker honey bees produce the beeswax.
The gland on the base of the bee’s abdomen is where the wax is buried and from here the wax
is moulded into six sided cells which are filled with honey and then capped with extra
wax.1Within the Pharmaceutical sector, beeswax forms only a tiny part of both the final
product and manufacturing process. The purifying of beeswax for distribution is not very
common at the moment. There is no additional component for beeswax within the cosmetics
and pharmaceutical industries. Most beekeepers use what they produce especially those
beekeepers using frame hive technology who are their own best customers. In many
situations in industry the beeswax is supplemented for by using synthetic waxes mainly due
to cost and also because their own processing promises better quality control. By using these
waxes the manufacturers accept the compromises in quality of the synthetic waxes over the
beeswax. In the early 1980’s in the USA beeswax prices for imports went above US$4/kg but
are currently changing between US$2.10 and 3.00/kg for light coloured wax sometimes
reaching US$6 - 7/kg. However darker wax is approximately 10-20% cheaper. Similarly to
honey prices, prices for beeswax may differ substantially from place to place.
The definition of a skin cream is to provide moisture to the skin and for replacing certain oils
of the skin. The basic formulation of a cream contains oil (mineral or vegetable), water and a
wax in order for the formulation to have a creamy appearance and also to allow equal
distribution of the water. As oils or wax do not mix with water an emulsifier is added, usually
borax. The function of the emulsifier is to change the acids in the wax into soap which in turn
will then mix with the water to give a creamy texture. The ingredients of any formulation can
change but it is advised that no more than 6.8% of borax on the weight of the wax should be
used. It should be noted that when making a cream one should always remember that if too
much borax is added the cream will have a rough texture as borax is not very soluble.2

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1.1.0 UV Filters
The existence of UV filters in skin care and cosmetic products represents a key advantage
that cosmetics can provide to consumers. Ultraviolet light (UV) carry’s many hazards which
are well known. Every year within the United States it is estimated that the occurrence of
non-melanoma skin cancer cases exceeds one million. There are two particular reasons that
account for most of the age associated changes in skin appearance, these are photo aging and
UV induced. UV radiation can damage the skin in two ways firstly by direct effects on DNA
and secondly by indirect effects on the skins immune system. Sunscreens can prevent the
formation of squamous cell carcinomas of the skin in animal models. Sunscreens have also
shown to decrease the number of actinic or precancerous keratosis and solar elatosis when
used regularly. In an Australian study it was shown that the daily use of sunscreens on the
face and hands of people reduced the total incidence of squamous cell carcinoma of the skin.
The definition of an Immunosuppressant involves an act that reduces the activation or
efficacy of the immune system. It has been documented that the use of sunscreens can also
prevent this immunosuppression taking place. Studies carried out in the area of double blind
photo aging have demonstrated steady improvements in the untreated control groups partly
because of the use of sunscreens by all study subjects. However with regards to melanoma
the effect of sunscreen use is less clear. A meta-analysis of population based studies of
population based case control studies found no effect of sunscreen use on risk for melanoma.
Nonetheless observational studies have indicated that intermittent or intense sun exposure
contributes to the increased risk for melanoma. This observation would support the theory
that avoiding sunburn specifically in childhood may possibly lessen the risk of developing
melanoma. The formulation of cosmetics is an ever expanding list of options of active
sunscreen ingredients for integration into a wide range of cosmetic formulations. Although
there a large number of these sunscreen ingredients, choice is limited by the regulatory
parameters that are in place in the country in which the product is to be advertised. 3

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1.1.1 History of UV Filters
In the 1890s acidified quinine sulphate was proposed for use as a chemical sunscreen. Paul G.
Unna (1850–1929) was a dermatologist who was the first to associate UV exposure and
precancerous skin changes seen in sailors (Unna 1894). At the start of the 20th century Unna
established aesculin which is a chestnut extract that was being used in traditional medicine
for many years. He noticed that this extract was far more effective as acidified quinine
sulphate. In 1928 two UV filters benzyl salicylate and benzyl cinnamate were the first
incorporated into sun cream products in the United States. Phenyl salicylate was another UV
filter that was used in an Australian product in the early 1930s. 4
In 1943 4-aminobenzoic acid (PABA) was patented which lead to the expansion of PABA
derivative UV filters. Throughout World War II the US military used a UV filter called red
veterinary petrolatum. This stimulated the development of additional UV filters in the
subsequent period after the war. During the 1970s, increased interest in commercial
sunscreen products develop which led to enhancements and customer acceptance of these
products over the next twenty years. As people became more and more aware as to the
hazards of UVR, higher sun protection factor (SPF) products became the standard. Over the
past ten years consumer products containing UV filters such as colour cosmetics,
moisturizers and hair care products have become more predominant in the daily lives of
people. In the last few years there has been a greater interest in broad spectrum sunscreen UV
protection throughout the entire UVA range due to concerns relating to the capability of
sunscreen protection for the prevention of photo aging and melanoma.5
There are two forms of UVR that reach the earth’s surface UVB (290-320nm) and UVA
(320-400nm). UVA can then be divided into UVA I (340-400nm) i.e. far UVA and UVA II
(320-340nm) i.e. near UVA. The definition of the sun protection factor (SPF) is the dose of
UVR needed to give one minimal erythema dose (MED) on protected skin after application
of 2 mg/cm of product divided by the UVR to produce 1 MED on unprotected skin. With
regards to the area of water resistance products can be tested for between forty to eighty
minutes in order to determine see if the product maintains its SPF level. For example if they
are very water resistant/waterproof or just water resistant. A sunscreen which provides both
UVB and UVA protection will possess a broad or full spectrum. This would fall into the
UVA I and UVA II regions. 6

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1.1.2 Regulation of UV Filters
United States
Sunscreen products in the United States are regulated by the FDA as over-the-counter (OTC)
drugs. The final monograph for sunscreen drug products for OTC human use established the
conditions for safety, efficacy, and labelling of these products. A recently proposed
amendment further elaborates on UVB (SPF) and UVA testing and labelling. As active
ingredients in drug products, they are listed by their United States Adopted Names (USAN).
There are 16 approved sunscreen ingredients (Table 1.0). All permitted UV filters can be
used with any other permitted filters except avobenzone. The FDA regulates sunscreen
products in the United States as over the counter drugs (OTC). Conditions for safety, efficacy
and labelling of various products have established a final monograph for sunscreen drug
products for OTC human use. UVB (SPF) and UVA have recently seen amendments further
elaborating on testing and labelling. The United States Adopted Names (USAN) has listed
both UVB and UVA as active ingredients in drug products. Sixteen sunscreen ingredients
have been approved for use in the US by the FDA. Avobenzone is the only filter that is not
permitted to be used with other permitted filters.
Avobenzone can also not be used with PABA, octyl dimethyl PABA, meridamate and
titanium dioxide (TiO). The maximum concentrations allowed are provided. Minimum
concentrations were substituted only if the concentration of each active ingredient was
sufficient enough to give at least an SFP rating of no less than 2 to a finished product. A
sunscreen product must have a minimum SPF of not less than the number of active sunscreen
ingredients used in combination multiplied by 2. Recent amendments for sunscreen products
have prohibited the term ‘sun block’ but the term ‘UVB’ is to be incorporated before the term
SPF on the main product display panel. Not only are the active ingredients of the UV filters
to be included in newer labelling requirements but also is the concentration of the UV filter
present in the product. The FDA have proposed a combination of spectrophotometric (in
vitro) and clinical (in vivo) testing measures to permit for a non-numerical UVA protection
four star rating system, with four stars being the highest and one being the lowest. The main
reason for these planned changes is to correct the inadequacies of any particular UVA rating
system. The persistent pigment-darkening (PPD) method is to be used for the in vivo study.
The Boots adaptation of the Diffey/Robson method has been suggested for in vitro testing in
the most recent amendment.

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Table 1.0 Lists of the UV filters that were analysed in this project and their maximum
concentrations allowed in cosmetic products in the U.S.A. 7
UV filter Maximum concentration %
Butyl methoxydibenzoylmethane (BDM) 3
Benzophenone 3 (BZ3) 6
Octylcrylene (OCR) 10
4-Methylbenzildene Camphor (MBC) Not allowed
Octyl methoxycinnamate (OMC) 7.5
Octyl salicylate (OS) 5
Octyl dimethyl PABA 8
Europe
In Europe, sunscreen products are considered to be cosmetics, their function being to protect
the skin from sunburn. Definitions and lists of the UV filters that cosmetic products are
permitted to contain are provided in the third amendment of the European Economic
Community (EEC) Directive. Table 1.1 lists the UV filters that are fully permitted and there
maximum allowable concentration levels in cosmetics. The European Union has approved
certain ingredients that are not available in the United States. Titanium monoxide has been
added to the approved list by the EU. Zinc oxide is not on the allowed list by the EU but it
can still be used as a cosmetic ingredient. A more recent commission directive 2006/647/EC
offers additional assistance on UVA/UVB efficacy claims. The PPD method is clinically
recommended. With regards to in vitro testing, the critical wavelength method is to be used in
contrast to the Boots adaptation recommended by the FDA.

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concentrations allowed in cosmetic products in the E.U.
4-Methylbenzildene Camphor (MBC) 4
Octyl methoxycinnamate (OMC) 10
Australia
In Australia sunscreens are licensed as therapeutic goods. In 1993 Australia and New Zealand
jointly published the latest edition of Australian standard 2604. Sunscreens products are
categorised as primary or secondary depending if there main function is to protect from UVR
or a product with a sole cosmetic purpose. SPF of 30 denotes the maximum designation in
cosmetic products anything greater than 30 is not permitted. Australia has its own approved
list of names for active sunscreen ingredients which is pretty much the same as the FDA lists
of names with only a limited number of differences.
concentrations allowed in cosmetic products in Australia.
4-Methylbenzildene Camphor (MBC) 4

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Other Countries
The EEC directive is followed by most non-EEC countries. The U.S. trend is followed some
countries with their provisions. Similar to Europe, Japan also classifies sunscreens as
cosmetics. Regulations need to be considered for each individual country prior to the
implementation of UV filters into a sunscreen product that is to be advertised in a given
region. 8
concentrations allowed in cosmetic products in the Japan.
4-Methylbenzildene Camphor (MBC) Not allowed
On the 27 July 1976 the Cosmetics Directive 76/768/EEC was published. What this directive
exactly permits is for cosmetic products within the European Economic Area to circulate
freely and guarantees their safety for use. The definition of a cosmetic product is any
substance that can make contact with external parts of the human body or with the teeth with
the idea of cleaning them, changing their appearance, correcting smells, protecting them or
preserving their good condition. When used under normal conditions these products must not
be harmful to human health.
There are certain standards for cosmetic products set out by the cosmetic directive that must
be met before they can be used in the European Economic Area, for example substances that
cannot be included in the formula of the products, requirements for labelling and packaging,
rules for market surveillance and notification to the competent authority of each member state
and laws relating to animal testing. Since it originated, the European Parliament and the
European Council have amended the Cosmetic Directive 55 times in order to maintain it with
the changing cosmetic market.

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Other EU directives and regulations which may apply to cosmetic products are the 94/62/EC
Packaging and Labelling Directive, the (EC) REACH 1907/2006 Directive, permitted and
restricted ingredients, cosmetic guidance documents and European Court of Justice Rulings.
There are 27 EU countries in which the Cosmetic Directive is valid as well as the EFTA/EEA
states. The Cosmetics Directive 76/768/EEC has outlined certain substances that cannot be
used in the formulation of a cosmetic product, and also it states a list of substances that
cosmetic products can only contain under certain restrictions.
The directive also has certain requirements labeling. The packaging and containers must
demonstrate the following information:
 The name and address or registered office of the manufacturer or person responsible
for the marketing of the cosmetic product
 The weight or volume of the product at the time of packaging
 A ‘’Best used before’’ date for products with a durability of less than 30 months, and
for products with a durability of more than 30 months the period of time for which the
product can be used without causing harm to the consumer after the product has been
opened.
 Precautions for use
 An identification number
 The function of the product
 A list of ingredients in descending order. 9

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1.1.3 Mechanism of Action
The mechanism of action of UV filters by tradition has been divided into chemical absorber
and physical blockers. Aromatic compounds conjugated with a carbonyl group are mostly
referred to as chemical sunscreens. High intensity UV rays are absorbed by these chemicals
which then i.e. the chemicals get excited to a higher energy state. The energy that is lost
returns to the ground state as longer lower energy wavelengths. Modern sunscreen chemicals
have evolved over time and denote a prototype study in the use of structure-activity
relationships to design new active ingredients.
The study of structure-activity relationships has been well revised throughout the cosmetic
world. Physical blockers in the arrangement of newer micro sized forms may also work in
part by absorption. UV filters can be categorized into two sets, inorganic or physical filters
and organic or chemical filters. Inorganic filters function by reflecting or scattering the UV
light while the organic filters absorb it. Titanium dioxide is the only physical filter which is
approved for use by the EU Cosmetic Directive. There are at the moment twenty seven UV
filters permitted by the EU Cosmetic Directive for commercial use.10

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1.1.4 Developments in UV Filter Technology
Sun protection technology has developed rapidly over the last 30 years. The damage that UV
radiation can cause to the skin has become more prevalent in the public domain and doctor’s
knowledge of the damage that the mechanisms of these UV filters can cause has increased.
This increased knowledge of UV filters has stimulated many new innovations in effective UV
protection products. The development of novel sunscreens continues today with more
sunscreen products being made and more successful ways to deliver these products onto the
skin being investigated.
The ideal sunscreen molecule should also possess the following characteristics:
 Safe to use on skin i.e. non-toxic, non-irritating and wont clog pores (non-
comedogenic).
 Does not penetrate into or through the skin.
 Good solubility in cosmetic moisturizers.
 Photostable i.e. does not breakdown when exposure to UV light.
 UV spectral profile not significantly affected by solvents.
 Adaptable with other cosmetic ingredients.
 Does not give off any smells or colours in the final product or on skin.
 Chemically stable.
 Compatible with most packaging materials.
UV filters are most commonly defective in at least one of these areas. “For example,
ethylhexyl methoxycinnamate (octinoxate) and butyl methoxydibenzoylmethane (avo-

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benzone) are photolabile; avobenzone can also be difficult to solubilize, as can
benzophenone-3 (oxybenzone)”11. New advances in organic UV filters have a tendency to
target these weaknesses or to improve the UV absorption effectiveness. To improve UV
absorption efficacy the extinction coefficient may possibly have to be increased or the
spectral coverage would have to be broadened, specifically since awareness of the skin
damage that can be caused by UVA radiation has grown. This growth has prompted the
development of numerous new UV filters over the past few years. Double, triple or extended
chromophores are a common design feature in new UV absorber molecules. The outcome of
these design features is UV filters that are more effective, less influenced by light
(photostable) and display minimal levels of skin penetration due to the size of the molecules.
The number of inorganic sunscreens ingredients is small, only zinc oxide (ZnO) and titanium
dioxide (TiO2) are of commercial significance and these active ingredients are no longer
considered new technologies. In Europe 60% of commercial sunscreen products contain TiO2
and also in various parts of the world, for example Japan where the percentage is even higher.
There have been developments in coating, ease of use and compatibility with other
ingredients and photostability of inorganic sunscreens. Also recent improvements in TiO2 and
ZnO particle size distribution have resulted in less transparency on the skin and no whitening
which was customarily associated with these products. Although there has been one recent
development in inorganic sunscreen technology that can be defined as new and this is the
development that transforms TiO2 from a simple UV filter into a multifunctional ingredient.12
In the areas of personal care and sun protection the use of more natural ingredients is a
growing market. There is only one main problem that is to find a truly natural sunscreen
ingredient. Natural mineral sources are contained within inorganic sunscreens and numerous
commercial sun care formulations only use inorganic filters. Such products have been
advertised as all natural i.e. contains natural minerals. Several botanical extracts have
demonstrated sun protective properties, by the benefit of containing antioxidants or chemical
species which can function as UV filters. Bobbin et al investigated the natural extracts and
their UV filter properties and established that some of the extracts possessed substantial UV
absorption but the extinction coefficients were quite low and in several cases the absorption
maxima lien in the UVC wavelengths at too small a wavelength to be of concrete use to
sunscreen formulations. Kapsner et al and Epstein et al have also investigated botanical
extracts but at the moment none of these botanical ingredients have been approved for use as

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UV filters, and their fairly weak UV-absorbing effects would indicate that none are likely to
be pursued commercially for this purpose alone. A more likely use for natural extracts may
be by mechanisms which can protect the skin other than UV absorption.
The physical form of the sunscreen product is another area of sun protection which has been
the topic of substantial review in the last few years. The main reason for this particular
development has been ease of use which is hoped will result in greater customer compliance.
Traditionally sunscreen products have been designed as creams and ointments submission of
such products is deemed messy and timely. With children and teenagers being the most
important target groups this can prove problematic. The European market in recent years has
seen the development of spray able emulsion systems while in the U.S. aerosol sprays now
possess a large share in the U.S. market. Advances in emulsion technology have led to
developments in emulsion sprays. At first sunscreen products centred on emulsion sprays had
very low SPF factors (5 or 10) but now since advances in emulsion technology products are
available with SPF factors of 30 or 50.
Sun protection technology has developed rapidly over the last twenty years. As the awareness
of the dangers of the sun has increased as have the request for ever greater SPF values and
broad spectrum protection products. These demands have given rise to the development of
new sunscreen ingredients and also more effective modes of delivery for these ingredients.
Most of these new ingredients have not received approval from the necessary regulatory
authorities and for this reason can still be regarded as emerging technologies, even though
they have been in existence for many years. Most of the modern UV filter technology is
focused on the delivery systems of the active ingredients so that they will be more proficient,
safer and more stable thus giving high protection factors without the use of extremely high
concentrations of UV filters. Novel formats for final products is been developed with the
intention of making sunscreen products more convenient in everyday life.

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1.2 Scope of High Performance Liquid Chromatography
In the mid 1970’s High Performance Liquid Chromatography (HPLC) was developed and by
the 1980’s HPLC was undoubtedly the most exact and sensitive method for separating
chemical mixtures. HPLC separation is attained by injecting the sample dissolved in the
solvent of choice into a stream of solvent being pumped into a column packed with a solid
separating material (stationary phase). The detector then detects the liquid sample.
High Performance Liquid Chromatography is the most extensively used of all of the
analytical separation techniques. This statement is further backed up by yearly sales of HPLC
tools approaching the one billion dollar mark. The explanations as to why HPLC is so
popular are; its flexibility to accurate quantitative analysis, its sensitivity, its appropriateness
for separating thermally fragile or non-volatile species and its extensive applicability to
substances that are of significant interest to science, to the public and to industries. Examples
of such material’s consist of; steroids, antibiotics, pesticides, carbohydrates, hydrocarbons,
amino acids, nucleic acids, proteins, multi-organic species and a range of inorganic
substances. 13
Numerous liquid chromatography techniques tend to be linked with regards to their areas of
applications. Exclusion chromatography is often used for solutes having molecular weights
larger than 10,000 however reverse phase partition chromatography is now becoming
possible to handle such compounds. Ion exchange chromatography is widely used for lower
molecular weight ionic species. Partition methods are best suited for small polar but non-
ionic species. To separate members of a homologous series partition methods are commonly
used. Separation of non-polar species, structural isomers and compound periods such as
aliphatic hydrocarbons from aliphatic alcohols adsorption chromatography is regularly
selected.
Adsorption chromatography with solid stationary phases has been largely replaced by normal
phase chromatography mainly due to the problems with retention reproducibility and
irreversible adsorption. 14

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Figure 1.2 Schematic Diagram of a HPLC15

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1.2.0 Eluents in HPLC
The development of a HPLC separation usually focuses on finding the right mobile phase
solvent mixture. While there are many solvents to choose from only a few have all the
desired properties. Solvents possess many physical properties and these consist of:
1) Viscosity: low viscosity solvents lower the back pressure to achieve a given flow rate.
2) Boiling point: low boiling point facilitates solvent removal from collected fractions.
3) Detector compatibility: solvent should not interfere with the operation of the detector.
For UV detection the solvent should have a low absorbance of the chosen wavelength.
For the refractive index detector the refractive index of the solvent should not be near
that of the sample.
4) Safety: solvents with low toxicity should be preferred for both disposal and safety
reasons.
To be useful with UV detection the solvent has to have a lower cut off point than any of the
sample components.
Table 1.2.0.1 Solvent properties for the solvents that were used in this project.
Solvent Boiling Point Viscosity UV cutoff Refractive index
Ethanol 78.3 1.21 205-210 1.361
Acetonitrile 82 0.38 190 1.344
Eluents are usually a mixture of two or more solvents. The composition of the solvents
influences both retention and selectivity. Retention is influenced by mixing both strong and
weak solvents.
Table 1.2.0.2 Solvent strength for reverse phase chromatography.

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Solvent Strength
Water 0
Methanol 3.0
Acetone 3.4
Ethanol 3.6
Propanol 4.2
Tetrahydrofuran 4.4
In this example water is the weak solvent and the organics are the strong solvents. From this
table a high ratio of water would result in an extended run time whereas a high concentration
of tetrahydrofuran would reduce the run time considerably. This is due to the greater
interaction of the strong solvent with the analytes resulting in more rapid movements through
the stationary phase.

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1.2.1 Retention Time (tr)
The retention time tr is the time required to elute a peak, retention is often expressed in terms
of capacity factor k-1.
Defined as k-1 = tr - to or in some cases V2 – V0
to V0
Where to is the time required to elute an un-retained species and tr represents the species of
interest.
The longer a component is retained by the column the greater the capacity factor. The
capacity factor is a measure of how well the sample is retained by the column during an
isocratic separation. The capacity factor is affected by the column packaging and the elution
conditions.
Kd is known as the distribution constant which measures the equilibrium distribution of a
species between the stationary and mobile phases. K-1 is constant for a particular solute and
depends only on kd, Vmwhich is the volume of the mobile phase and Vs is the volume of the
stationary phase in the column.
k-1 = kd Vs Vm
Is the relationship between the capacity factor, the distribution constant kd and the volumes
of the two phases Vs and Vm.
Retention time is dependent on elution flow rate. A more fundamental retention parameter is
retention volume Vr. Retention volume is the volume of the eluent that has passed through the
column at the retention time. The retention volume of a non-retained component is equal to
Vn the mobile phase in the column.
Retention volume is the product of the retention time and flow rate F.16

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1.2.2 HPLC Pumps
Liquid chromatographic pumps must be capable of generating pressures of up to 414 bar,
have pulse free output, have flow ranging from 0.1 to 10ml/min, flow reproducibility’s of
0.5% relative or better and resistance to corrosion by a variety of solvents. There are two
major types of pumps that are used in HPLC these being the screw driven type and the
reciprocating pump. Most modern commercial chromatographs employ reciprocating pumps.
Types of HPLC Pumps
Reciprocating Pumps
In reciprocating pumps the solvent is pumped in a back and forth motion by a mechanical
piston. The flow of the solvent in and out of the cylinder is controlled by two ball check
valves which open and close alternately. The piston is in direct contact with solvent. The
disadvantage of reciprocating pumps is that a pulsed flow is produced which in turn produces
pulses which appear as baseline noise on the chromatogram. Current HPLC instruments
employ elliptical cams or dual pump heads to reduce such pulsations. There are many
advantages of reciprocating pumps such as:
 Small internal volumes ( 35-400µL)
 Large solvent capacities
 Flexibility to gradient elution
 High output pressure ( up to 690 bar)
 Constant flow rates (free from solvent viscosity and column back pressure)
Displacement Pumps
Large syringe like chambers equipped with a plunger activated by a screw-driven mechanism
powered by a stepping motor is usually what a displacement pump is made up of. Like
reciprocating pumps displacement pumps also produce a flow that tends to be free from
column back pressure and solvent viscosity. Also the output that is generated by displacement
pumps is pulse free. Disadvantages of this pump include its inadequate solvent capacity
(approx 250ml) and the problems encountered with solvent change.17

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Figure 1.2.2 A reciprocating pump for HPLC.18

Page | 24
1.2.3 Sample Injection
The most popular method of sample introduction in HPLC is based on sampling loops. These
accessories are an essential part of HPLC instruments and have substitution loops that permit
a choice of sample sizes from 1µL to 100µL. Types of these sampling loops allow the
introduction of samples at pressures up to 483 bars. Modern chromatograms are sold with
auto-injectors. These systems inject samples into the HPLC from glass vials on a sample
carousel. A number of these systems have controlled temperature environments that permit
sample storage and derivatization reactions preceding the injection. An unattended injection
is the norm is today’s HPLC instruments as these injections are programmable.19
Figure 1.2.3 A sampling loop for HPLC.20

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1.2.4 Columns
In HPLC the column is one of the most essential components because the separation of the
sample components is achieved when those components pass through the column.21There are
many types of columns such as reverse phase, normal phase, ion-exchange, size-exclusion
and adsorption columns. A Thermo Hypersil C18 BDS column was used in this project.
Thermo Scientific Hypersil BDS columns were first introduced in 1989 and have since
gained a status as one of the most robust, reproducible and dependable HPLC columns
available. The main benefits of the base deactivated silica based (BDS) columns are:
 Reduced Silanol interactions.
 Reduced peak tailing.
 Reduced need for mobile phase additives.
 Excellent peak symmetry.
 Long column lifetimes.
 Improved performance with basic, neutral and acidic compounds.22
Packaging Characteristics
Many reverse phase chromatography (RPC) columns undergo a bonding step where
trimethylchlorosilane is added and this technique is known as end capping.
Particle size: The diameter for HPLC column packing can range between 3-20 microns.
When the particles are small there is less pressure required to pass the samples through the
column. A particle size of 3-10 microns is used for analytical work and bigger than 10
microns is used for preparative work.
Particle shape: Spherical and irregular are the two different shapes that particles are available
for HPLC. There is less pressure needed when spherical shapes are used for eluent velocity
and this gives a more permeable packing structure.
Porosity: Porous or pellicular are the two forms of packing for a column. Approximately 50%
of the volume of a particle is open pore volume. The internal surface of the porous particle
can be up to 400m2/g. Pellicular packing contains a porous active ingredient on the surface of
a solid inert core. Greater permeability is achieved with this type of packing than with porous

Page | 26
packing. However pellicular packing’s have less surface area, smaller ability to retain
samples and can only manage small loads.
Surface chemistry: The retention and selectivity characteristic for packing materials is
determined by the surface functional groups. The solutes can react with these functional by
intermolecular forces such as hydrogen bonding.23

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1.2.5 HPLC Detectors
The characteristics of an ideal detector include:
 Good sensitivity
 Good stability and reproducibility
 Short response time free from flow rate
 High consistency and ease of use
 Good response to solutes
 Non-destructive
Types of Detectors
UV Absorption Detectors with Filters
The first absorption detectors were filter photometers with a mercury lamp as the light
source. Most frequently the intense wavelength at 254nm is isolated by the filters. In certain
instruments wavelengths at 250, 313, 334 and 365nm can also be employed. Therefore these
detectors are restricted to solutes that absorb at one of these wavelengths. Deuterium or
tungsten filament lamps can also be used to detect absorbing species as they elute from the
column. Certain instruments are equipped with filter wheels containing a number of filters
that can be quickly switched to detect various species as they elute off the column. Modern
instruments have too a large degree replaced by scanning and diode array spectrometers.
Photodiode Array Detector
The most dominant UV spectrophotometric detector is array based instruments. These
detectors allow for the collection of an entire spectrum in roughly one second. Therefore
spectral data for each chromatographic peak can be collected and stored as it comes off the
column. This is very useful identification of species and for choosing the necessary
conditions for quantitative determination.
Fluorescence Detectors
Fluorescence detectors for HPLC are comparable in design to fluorometers and
spectrofluorometers. The main advantage of fluorescence detectors is their high sensitivity
which is approximately 10-10,000 greater than the UV/Vis detector. This particular
advantage has been employed for HPLC separation and identification of samples that

Page | 28
fluoresce. Compounds that fluoresce are constantly being encountered in the pharmaceutical
industry.24

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1.2.6 HPLC Modes
Adsorption and bonded phase separations are best described by the terms normal phase and
reverse phase. The definition of normal phase is the polarity of the stationary phase is greater
than that of the mobile phase which is what occurs for example when silica is used in
adsorption chromatography. The definition of reverse phase is the polarity of the stationary
phase is smaller than that of the mobile phase which is what happens polar mobile phases and
hydrocarbon type bonded mobile phases. For both normal and reverse phase solutes are
eluted in order of polarity with normal phase least polar first and with reverse phase most
polar first. The retention times of the solutes can be altered by changing the polarity of the
mobile phase or of the stationary phase. The pH of the mobile phase is a key factor in terms
of retention and selectivity for ionisable solutes.
Reverse phase operation with bonded phases has many advantages which had led to its
popularity:
 Very broad scope which permits samples with extensive ranges of polarity to be
separated.
 Uses environmentally friendly and fairly cheap mobile phases.
 Can be used for the separation of ionisable or ionic compounds.
 Easy, fast and more precise than other HPLC modes.25
However reverse phase operation has its disadvantages:
 Stable columns can only be maintained over a pH range of between 3-8.
 Tailing, high retention times and non-reproducible results can often occur due to the
presence of unreacted silanol groups on the silica surface.

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Table 1.2.6.1 Characteristics of reverse and normal phase chromatography26
Normal phase Reverse phase
Stationary phase High Low
Mobile phase polarity Low-medium Medium-high
Typical mobile phase Heptane/CHCl3 CH3OH/H2O
Order of elution Least polar first Most polar first
To increase retention time of
solutes
Decrease mobile phase
polarity
Increase mobile phase
polarity

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1.2.7 Quantitative Analysis
Quantitative analysis involves measuring how much of a substance or substances is present in
a sample. Quantitative analysis is very much linked to the chromatographic apparatus
especially the detector and the injection system. Firstly the sample must be placed on the
column by the injection system and there after the detector must have a linear response that is
known and defined by its response index. The concentration of the linear range should lie
between 0.97 and 1.03 which would demonstrate good accuracy with regards to the response
index of the detector. An alternative measurement which can be used to measure the quantity
of a component present is peak height provided that the separation is highly reproducible.
Peak area is the most common method used for quantitative measurement as it is more
precise. The major application of chromatography techniques is probably quantitative
analysis.27
In this project HPLC was used to determine the quantity of the UV filters in the samples that
were made up. The sample was prepared by dissolving a known amount in ethanol and was
subsequently filtered and analyzed. A range of standards of UV filters were prepared and ran
on the HPLC instrument. From the results a graph was plotted of the standards peak area
versus the concentration. In order to find the concentration of the sample the peak area for the
sample was filled in for y on the equation of the line and the corresponding concentration was
found. A calculation was carried out using the original amount weighed out and the
concentration of the UV filter present in the sample was found using the equation of the line.
It was then possible to find out the percentage recovery of the UV filters.

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2.0 Literature review

Page | 33
Schakel et al determined of sixteen UV filters in sun care formulations by High-performance
liquid chromatography. The UV filters that were analysed were; 4-Aminobenzoic acid
(PABA), Homosalate (HMS), Benzophenone-3 (BENZ-3), 2-Phenylbenzimidazole-5-
sulfonic acid (PBSA), Terephthalidene dicamphor sulfonic acid (TDSA), 4-tert-Butyl-4-
methoxy dibenzoylmethane (BMDBM), Octocrylene (OC), 2-Ethylhexyl-4-
methoxycinnamate (EMC), Isoamyl-p-methoxycinnamate (IMC), Ethylhexyltriazone (ET),
Drometrizole trisiloxane (DTS), Diethylhexyl butamido triazone (DBT), 3-(4-
Methylbenzyliden) camphor (MBC), 2-Ethylhexylsalicylate (ES), 2-Ethylhexyl-4-
dimethylaminobenzoate (ED-PABA) and Benzophenone-4 (BENZ-4). The HPLC instrument
that was used was an Agilent liquid chromatographic system equipped with a binary pump,
an injector with variable loop and a DAD was used. The UV filters were analysed by using a
5µm RP C18 column and the mobile phase that was employed by Schakel et al was a gradient
of ethanol-aqueous acetate buffer containing EDTA. The EDTA was added to the mobile
phase as a modifier to perform the HPLC separation of the UV filters as certain filters such as
BMDBM, EMC, ED-PABA, ES and HMS are difficult to separate. The majority of
extractions that are carried out in cosmetics are performed using ethanol or methanol with a
low pH and /or a high temperature.
In this study the extraction was carried out at a high temperature (60ºC) followed by
sonication with the ethanol as the extraction solvent and also Twen 80 was used to break
down the emulsion. 313nm and 360nm were the wavelengths for UV detection that were used
in this study. It was found that a temperature of 28ºC resulted in optimum separation of the
filters resulting in good chromatographic peaks especially for the BMDBM. During
developing their method Schakel et al noticed that the performance of the column changed.
The chromatographic performance of the BMDBM changed as peak tailing of the BMDBM
occurred. This was due to the fact that the BDDBM and ES both eluted at the same time so
quantification proved difficult. By adding the EDTA resulted in good chromatographic peak
shape. As mentioned the BMDBM peak was giving problems so what Schakel et al found
was that the higher the concentration of EDTA, the better the peak shape of BMDBM
became. A high concentration of EDTA would affect the performance of the pump so they
decided to use 0.2mM EDTA.28
Salvador et al carried out a critical survey on UV filters determination in sunscreens. They
reviewed many literature journals in the area of UV filters in cosmetic science. They found

Page | 34
many papers such as Gasparro et al who investigated the safety and efficacy of sunscreens
has been investigated by, likewise Nohynek and Schaefer published a similar paper detailing
the advantages and disadvantages of the use of UV filters. Lowe et al published a book
reviewing the chemistry and other aspects of sunscreens. Granager and Brown published a
very interesting article on the chemistry of UV filters and the use of liquid chromatography as
analysis. Chromatographic techniques such as thin layer chromatography were employed for
identification purposes. Eiden et al employed such a technique to separate methyl
phenylbenzoxazole. They did this firstly by identification by measuring its IR and UV spectra
and secondly by quantification by both gravimetric and photometric methods. They also
identified cinoxcate in a similar manner. Liem and Hilderink suggested TLC combined with
UV absorption spectrometry for the quantification of 50 UV filters, but they only found 24 in
the 197 analysed samples.
The research group of Sherma and co-workers also quantitatively determined UV filters by
High Performance Liquid Chromatography (HPLC) using a densitometric detector. TLC has
also been used for the isolation of compounds that are banned in cosmetics, although they
may be present as contaminants of approved UV filter. Schmitz-Masseet al separated glyceril
PABA and other esters of p-aminobenzoic acid by using TLC and their identification was
carried out by NMR. Paulis was first to publish a paper for UV determination using gas
chromatography (GC) in 1972. Paulis characterized cinnamates and salicylates in suntan
preparationsby GC. GC has not been used much for the determination of UV filters as in GC
the compound must undergo both volatilisation and thermostability and the majority of the
UV filters have fairly high boiling points. The main reason as to why HPLC is preferred to
because it can deal with low-volatile compounds. Solvents usually employed in
chromatography have been water, acetonitrile (MeCN), methanol (MeOH) and
tetrahydrofurane (THF) or mixtures of them, either with isocratic or gradient phases. The
authors of this review proposed several methods in which mixtures of ethanol (EtOH) and
water were used as mobile phase, thus eluding the use of more hazardous organic solvents.
Use of hydroxypropyl-β-cyclodextrine (HP-β-CD) has been used as a mobile phase modifier
in order to improve the resolution between different analytes. Gagliardi et al used mobile
phase modifiers such as per-chlorate and tetramethylammoniun chloride (TAC) in order to
improve resolution. Separations are usually done on reversed-phase (RP) columns which are
preferred over normal phase columns.

Page | 35
Other chromatographic techniques reviewed by Salvador et al have been used for UV filter
determination. These include a technique used by Broadbent et al, who used supercritical
fluid chromatography coupled with mass spectrometry detector (SFC–MS) to study the
photodegradation products formed on ultraviolet irradiation of EMC. Tomasella et al
developed a method based on micellar liquid chromatography (MLC) for the determination of
BZ3, EDP and EMC in sunscreen mixtures. Tomasella et al used sodium dodecyl sulfate
(SDS) as a surfactant and a mobile phase of 0.3% triethylamine and isopropanol. Pietta et al
also employed a technique called micellar electrokinetic chromatography (MEKC) to
determine five filters. They achieved this by using a using a phosphate buffer of pH 7
containing 30mM SDS and 2.5%MeCN. Klampf et al came up with a method based on
MEKC by using a mixture of the surfactants Brij 35 and SDS for the determination of nine
UV filters, this method was later applied to the analysis of commercial sunscreen products.
Derivative ultraviolet spectrometry (DMS) was employed by Scurei and Oprea to
quantitatively determine UV filters such as EMC and ES in cosmetic formulations. Azevedo
et al also used DMS for quantitative analysis of two UV filters namely PBS and BZ4,
however they used different sample preparations for each filter. Salvador et al used DMS to
determine EMC and BZ3 by a conventional method and also by a flow injection method.
Salvador et al method was used for the analysis of lotions and creams and a slight change was
made to the method for the analysis of lipsticks. PAB was also determined by SI analysis.
This was done calorimetrically by measuring its diazo derivative. Solid phase extraction was
used for the extraction of PBS and BZ4 in sunscreens and were analysed in the UV region.
Both PBS and BZ4 were retained in a strong anion exchanger micro column and were eluted
independently using a SI system. Townsend et al were the first to determine UV filters by
chemiluminescence. They developed a method to determine EDP by using a FI system that
enabled them to measure the produced chemiluminescence brought about by its reaction with
potassium permanganate in sulphuric acid medium. Lu et al used UV spectroscopy to for the
quantitative analysis of UV filters in sun creams by means of their UV spectra. NMR has also
been used by Moi et al for quantification and identification of BDM, Benzophenone-1, BZ3,
EDP and EMC. Raman spectroscopy was used by Cheng et al and Narayanan et al to
determine EMC and PAB. Atomic spectroscopy has been suggested for the determination of
the inorganic filter titanium dioxide (TiO2). Salvador et al did work on this by inductive
coupling plasma emission spectroscopy while Kawauchi and co-workers employed x-ray
fluorescence spectroscopy and Mason et al recommended this analysis be done by flame

Page | 36
atomic spectroscopy. Lambropoulou et al did analysis on water samples from swimming
pools for the presence of certain UV filters such as BZ3, EMC and ES. They did their
analysis by using a GC with a FID detector or GC-MS with a prior solid phase micro
extraction phase. Salvador et al established a method to solubilize TiO2 built on an acid
digestion in a micro wave oven and later carried out a fusion of the TiO2 with KHSO4 heating
with a Bunsen burner and dissolving the excess in concentrated sulphuric acid.29
Smyrniotakis et al developed and validated a method for the determination of four chemical
UV filters in sun care products by reverse phase High Performance Liquid Chromatography.
The aim of their work was to develop a method that was fast, sensitive and reproducible for
the separation of Tinosorb M, Eusolex OCR, Eusolex OS and Eusolex 2292 in sun care
formulations. The column that they used was a 5µm BDS RP C18. The mobile phase they
used consisted of a methanol: acetronitrile (90:10 v/v) with a flow rate of 1ml/min. The
injection they used was 20µl and analysis was carried out at room temperature at an
absorption wavelength of 313nm and the total run time was less than 15 minutes.
Smyrniotakis et al carried out recovery studies for each UV filters in four different kinds of
sun care formulations using the standard addition method. Solutions were subsequently
injected into the HPLC instrument. Calibration curves for each of the UV filters was
constructed and the corresponding peak areas for the samples were inserted into the equation
of the line to determine the amount of UV filter in the sample. Tinosorb M proved a
problematic peak to reduce its retention time, numerous solvent mixtures were tried to
achieve this. What was found was that without the presence of water in the mobile phase and
by increasing the methanol: acetonitrile ratio the retention time of Tinosorb M decreased
while the retention times of the other three compounds remained the same. Smyrniotakis et al
achieved what was stated in the title and by doing so developed a method which allowed
Tinosorb M to be analysed together with Eusolex OCR, Eusolex OS and Eusolex 2292 in a
single run.30
Salvador et al also worked on a method centred on reversed-phase liquid chromatography
with a gradient mobile phase using environmentally friendly solvents to determine 18 UV
filters (fat soluble and water soluble) in sunscreen products. As mentioned previously
Salvador et al did use different analytical methods to determine UV filters by liquid
chromatography or flow injection spectrometric techniques while evading the use of toxic
organic solvents. The only disadvantage to their method was that it could only be applied to a

Page | 37
small number of UV filters. In this new method developed by Salvador et al used a Hitachi
LC system equipped with a Hitachi L-7100 high-pressure pump and a Hitachi L-7420 UV–
Vis detector set at 313nm and a Li chrosphor RP-18 column column. They used a
thermostatic water bath to set the column temperature to 45ºC. The mobile phase of ethanol:
acetic acid was employed for the fat soluble filters and ethanol: sodium acetate buffer was
employed for the water soluble compounds. HS had two isomers (HS1 and HS2). HS was
determined using the isomer HS2 as it is more sensitive. One of the HS isomers (HS1) eluted
with BDM so BDM was determined at 360nm to avoid interference caused by the HS1
isomer. As the filters present lipstick and makeup samples are hard to filter out they were
sonicated to leach the analytes out of the sample which took several minutes leading to
increased sample preparation time. To separate out the most critical pair’s peaks the effect
was temperature (25-60ºC) was considered in order to reduce the run time. What resulted was
as temperature increased retention time decreased for IMC-MBC and BDM-OS however
separation for ODP–OMC and HS1–BDM separation got worse as temperature was
increased. They found that the best pH value was 4.75 for water soluble filters and 3 for fat
soluble filters in order to achieve the best separation for the filters. They also noticed that
when they flowed EDTA thought the column for 15 minutes between the separation of the fat
and water soluble peaks, this solved the problem of BDM tailing which was occuring due to
interference from the OS and HMS peaks. Their analysis time took 30 mins for the fat soluble
filters and 10 mins for the water soluble filters.31
Mary Wharton et al developed a method for the determination of seven UV filters found in
cosmetics and sunscreen products. The UV filters analysed were BZ3, MBC, ODP, OCR,
OMC, BDM and OS. An Agilent 1100 series HPLC dual pump system was used equipped
with a UV diode array detector set at 313nm and a thermo hypersil c18 BDS column was
used. It was decided to use a mobile phase of ethanol: 1 % acetic acid the same mobile phase
used by Salvador et al in separating out 18 UV filters. An isocratic mobile phase was initially
used by Wharton et al also the initial wavelength of 330nm gave a poor response for the OS
filter so it was decided to change the wavelength to 313nm and to change the injection
volume from 20µl to 10µl resulting in a better response for the OS filter. An improvement
that was noted from Wharton et al method in comparison to Salvador et al method was that
the OS peak for Wharton et al came off at 6 minutes whereas for Salvador et al it came off at
22 minutes. The wavelength of 313nm with the isocratic mobile phase for the seven UV
filters gave a run time of 10 mins with good resolution for the filters. Also by reducing the

Page | 38
injection volume peak tailing between the BDM and BZ3 was reduced resulting in better
sharper peaks and better resolution between the peaks. By increasing the organic content of
the mobile phase Wharton et al noticed a reduction in run time but with poor resolution. To
counteract this, a gradient elution was made which gave the best reproducible results. The
method was then validated.32
R. Rodil et al used a liquid-liquid extraction to determine nine UV filters i.e. BDM, IAMC, 4-
MBC, OC, BMBDM, EHMC, EHS and HMS from water samples. The total organic carbon
(TOC) of the wastewater was measured. A Hewlett-Packard 1100 series pump linked to an
1100 series auto sampler was used. HPLC separation was performed on a C8 column. The
flow rate that was used was 0.4ml/min and the mass spectrometer that was used was triple
stage quadrupole. They used Membrane-assisted liquid–liquid extraction to analyse the self-
made membrane bags that contained different polymer materials to see which different
polymers would absorb the analytes. In this study they examined several different types of
non-porous membranes to see which was the best suited material for the liquid–liquid micro-
extraction. Solvent desorption of the analytes took 30 mins and the extract was then analysed
by LC/MS. What they noticed was the different adsorption affinities of the UV filters for the
polymeric materials. Both polyethylene materials (LDPE, HDPE) adsorbed the analytes to a
larger extend than PTFE and PP material. They carried out numerous experiments to find the
best sample properties for the extraction of the compounds. They investigated the effect of
pH, salt, methanol to see if they had any bearing on the extraction process. They maximized
these parameters using a Box-Behnken Design. The addition of methanol had a positive
effect on the extraction yields while the addition of the NaCl had a negative effect for every
compound however the effect of pH on the compounds was inconsistent throughout. The best
conditions for the extraction process were found to be at low levels of NaCl and high levels
of MeOH. They also considered the effect of temperature in a range of 30°C to 50°C. From
studying the effect of temperature they observed that increasing the temperature from 30°C to
40°C notably improved the recovery while no increase was observed when it was put to
50°C. As a result they decided that 40°C was the optimum temperature for extraction. Both
2cm and 4cm bags filled with Propanol were used for extraction with the 4cm giving a
shorter equilibrium time for the more polar compounds i.e. BP3 however it was the 2cm bag
that was ultimately chosen due to its lower solvent volume requirement and also its lower
detection limits. Through there method R. Rodil et al achieved good % extraction efficiencies
ranging from 76%-101%. There were two levels at which the extraction efficiency was

Page | 39
measured which were 25 ng L−1 and 250 ng L−1. Both of these gave greater than 60% which
is quite similar to more time consuming methods such as solid phase extraction (SPE) and stir
bar sorptive extraction. The maximum concentrations of the nine UV filters were found in
raw and lake waste water samples.33
Gaspar et al developed a HPLC method to assess the influence of photostabilizers on
cosmetic formulations containing UV-filters and vitamins A and E. The UV filters analysed
by HPLC on a C18 column at a wavelength of 325nm for BP-3, OC, OMC, DEHN and
Vitamin A and 235nm for Vitamin E analysis. They employed a gradient mobile phase
consisting of 84% of methanol: isopropanol (55:45, v/v) as solvent A and 16% water as
solvent B. To determine the degree of separation of UV filters, vitamins and possible
interferents from the excipients they quantitatively analysed a control that contained no
vitamin or UV filter. They tried to optimize separation of the UV filters on the C18 column
through variation in the mobile phase. They tried an isocratic mobile phase of methanol:
water (88:12, v/v) which resulted in good separation of the UV filters (OMC, BP3 & OC)
under review in a run time of 27 mins. Due to polarity of the vitamins a gradient elution had
to be used. This gradient elution as mentioned above gave results in separating the UV filters
and vitamins. The HPLC results were then validated and all of the correlation coefficients
were beyond 0.999. By using the gradient elution over the isocratic elution retention times for
the UV filters and vitamins was reduced ultimately reducing analysis time. To determine
photostability the UV filters were subjected to UVA/UVB irradiation.
OMC recovery was not affected when any of the photostabilizers were used when in the
presence of vitamin A. When both vitamins were combined with the photostabilizers the
formulation comprising of (BP-3, OC, OMC, DEHN, Vit E and Vit A) was more
photounstable than the formulation of (BP-3, OC, OMC, Vit E and Vit A) with regards to
OMC and vitamin E recovery. In contrast when vitamin A alone was analysed in all
formulations the formulation comprising of (BP-3, OC, OMC, DEHN, Vit E and Vit A) was
the most photostable succeeded by the formulation of (BP-3, OC, OMC, Vit E and Vit
A).What Gaspar et al observed was that when OMC was combined with both vitamin A and
E photostabilizers influenced its stability. BTDC (benzotriazolyl dodecyl p-cresol) and
DEHN (diethylhexyl 2, 6-naphthalate) were the two photostabilizers under review and what
Gasper et al results showed was that BTDC proved to be the best photostabilizer to OMC
when combined with vitamins A and E.34

Page | 40
Rastogi et al worked on a method for the determination of twenty UV filters (18 permitted
and 2 non-permitted) by HPLC diode array detection. Their analysis was carried out by using
a PLRP-S HPLC column set to 25°C, using a gradient elution comprising of Solvent A:
acetonitrile, Solvent B: tetrahydrofuran, solvent C: buffer at a detection of between 240-
400nm. Analysis by HPLC was run for 45 mins which is a very long time e.g. for one
particular sample the retention times for three filters were 13, 27 & 29 mins. This is in
contrast to the other journals reviewed whose run times are considerably lower. An amount of
the sample was weighted out and methanol and 2M sulphuric acid were added and heated to
60°C until a uniform mixture was obtained. They determined the UV filters in the sample by
comparing the tr and spectrum of each peak of the sample solution with the tr and the spectra
of the standard through a spectral library. They used a max-plot chromatogram to achieve
this. They used two samples of know UV filter amounts to check for the correctness of their
method and what they found was that the filters in the products were identified by their
method. They also analysed for UV filters in lotions and creams using their method and
found the filters present in the lotions and creams were identified by their HPLC method.
They then validated their method for quantification of UV filters in sun care products.35
P.P. Zhang et al developed a method centered on dispersive liquid–liquid micro extraction
(DLLME) in combination with high performance liquid chromatography (HPLC) has been
for the analysis of UV filters. DLLME has attracted much attention due to its short extraction
time, low cost and ease of use. However centrifugation requires speed so it was here P.P.
Zhang et al made a homemade magnetic stirring device to assist the DLLME apparatus in
order to speed up the process. They used several UV filters from aqueous environments to
serve as model analytes. They carried out the extraction process in a binary system
comprising of the sample solution and the extract. This is where the magnetic stirrer can into
effect as P.P. Zhang et al used it to transfer the aqueous sample to the extract speeding up the
mass transfer process. As a result of no centrifugation step was needed. Their method was
carried out on a HPLC system with a binary high pressure pump and a photodiode array
detector using a C18 column and at a detection of 254nm. They looked at several parameters
in order to try and optimize the extraction process these being solvent volume, time, ionic
strength, stirring speed and sample pH. 1-Octanol was the chosen extraction solvent due to its
popularity in many liquid phase micro extraction procedures. Solvent volume is an important
part in DLLME as it can affect both efficiency and separation. Volumes ranging from 20µL-

Page | 41
60µL were investigated in this study and what was found was that 50µL was the optimum
extraction volume. Although separation at this volume resulted in poor unsymmetrical
chromatographic peaks so the volume was changed back to 40µL which gave more
symmetrical peaks. Extraction time was reviewed in the range of 2-50min and it was an
extraction time of 20mins which gave the maximum peak areas. In order to study the effect of
ion strength on the extraction procedure salt was added to the DLLME procedure and results
showed it had no effect. As the pH increased from 3-7 it was noticed by Zhang et al that peak
areas increased also. The stirring speed was investigated between 260-1300 rpm. What was
observed was that as the speed of stirring increased so did the efficiency of the extraction.
What they concluded from their investigation of these parameters was that the best extraction
was achieved at a stirring speed of 1300rpm, extraction time of 20min, pH of 7 and with no
salt addition. The lake samples was analysed under the above conditions and no analytes
were detected indicating the lake was free from these UV filters or they were present in
minute quantities n not sufficient to cause any harm to the wildlife. Limits of detection were
able to reach ng ml-1.36
A. Zenker et al preformed a method on the determination of nine organic UV-absorbing
compounds (UV filters) namely BP-1, BP-2, BP-3, BP-4, 4-DHB, Et-PABA, EHMC, 4-MBC
and 3-BC) in environmental samples. Tissue and water samples were investigated.
Quantification and identification of lipophilic UV filters was carried out using gas
chromatography electroionisation mass spectrometry and for mid-polar and polar compounds
liquid chromatography coupled to electrospray ionisation mass spectrometry was used. Nine
UV filters were analysed by an Agilent 110 HPLC system coupled to an LC/MS using a SB
C18 column at a column temperature of 30°C. A gradient elution of 90% water and 10%
acetonitrile was used for the analysis of the UV filters. For GC/MS analysis of the lipophilic
UV filters helium served as a carrier gas with a flow rate of 0.4ml/min and an injection
temperature of 70°C. The GC oven was maintained at a temperature of 40°C which resulted
in sharp peaks. The method developed by A. Zenker et al for the extraction of lipids and
proceeding clean up by HPLC or by GC/MS resulted in an accurate and sensitive technique
for UV filters in aqueous samples. A. Zenker method of a sample clean-up by HPLC resulted
in all nine UV filters being drawn well out from fish tissue with recoveries ranging from 72
to 102%. An additional improvement to Zenker et al method is that less animal tissue is
required for sample preparation compared to other procedure for UV filters in fish samples.37

Page | 43
RM PHARMACEUTICALS
Batch Book 1.0 Cosmetic Formulation
Page 1 of 4
Batch number. 04.04.06
Revision number. N/A Supersedes: N/A
Author: _______________ Ronan Mullane (QC Analyst)
Approved by: ___________ Mike Geary (QC Manager)
Chemical Requisition:
Chemical Amount
Sodium tetra borate (borax) 0.38g
Distilled water 20ml
Beeswax 7.0g
Mineral Oil 30ml
Butyl Methoxydibenzoylmethane (BDM) 2.0g
Benzophenone (BZ3) 3.0g
4-Methylbenzylidene (MBC) 1.5g
Description: Beeswax main components are palmitate, palmitoleate, hydroxypalmitate and
oleate esters of long-chain aliphatic alcohols, with the ratio of triacontanyl palmitate to
cerotic acid the two principal components being 6:1.
Systematic Name: Raw waxes
Molecular formula: approx. C15H31COOC30H61
Molecular Weight: 677.2259 g/mol
Health and Safety:
Refer to the relevant risk assessments.
1.0 Materials:
1.1 Reagents
 Beeswax
 Borax
 Distilled water
 Mineral oil
1.2 Equipment
 500ml glass beaker (x2)
 Analytical weighing balance
 Stirring/Heating mantle
 Magnetic stirring chips
 Magnetic rod

Page | 44
RM PHARMACEUTICALS
Page 2 of 4
 50ml graduated cylinder (x2)
 Plastic pipettes
2.0 Method
Step 1
Weight out accurately 7.0g ± 0.1g of beeswax on an analytical balance.
Actual weight: 7.0616g
Signed: ______________ (QC Manager) Date: ____________
Step 2
Weight out accurately 0.38g ± 0.1g of borax on an analytical balance.
Actual Weight: 0.3802g
Step 3
Weight out accurately 2.0g ± 0.1g of Butyl Methoxydibenzoylmethane (BDM) on an
analytical balance.
Step 4
Weight out accurately 1.5g ± 0.1g of 4-Methylbenzylidene (MBC) on an analytical balance.
Step 5
Weight out accurately 3.0g ± 0.1g of Benzophenone (BZ3) on an analytical balance.
Step 6
Accurately measure out 30ml of mineral oil using a 50ml graduated cylinder.
Actual Volume: 30ml

Page | 45
RM PHARMACEUTICALS
Page 3 of 4
Step 7
Accurately measure out 20ml of distilled water using a 50ml graduated cylinder.
Actual Volume: 20ml
Step 8
Place the beeswax and the mineral oil into a 500ml glass beaker and place the beaker on a
heating/stirring mantle.
Step 9
Place a stirring magnet in the 500ml beaker.
Step 10
Turn on the heating/stirring mantle to a revolution of 4000rpm until the beeswax is
completely melted and place the digital thermometer in the beaker.
Step 11
Heat the beeswax and mineral oil to 71ºC until melted.
Step 12
Place the water into a different 100ml beaker and place the beaker on the heating/stirring
mantle and place a digital thermometer in the beaker. Heat the water to 71ºC.
Step 13
When the water is heated to 71ºC add the borax into the beaker containing the water and
dissolve fully. Stir the contents of the beaker using a stirring magnet at a revolution of
4000rpm until the borax is dissolved fully.
Step 14
When the borax is dissolved fully add the weighted out BDM, MBC and BZ3 to the beaker.
Step 15
Add the water/borax, BDM, MBC and BZ3 into the beaker containing the oil/wax. Be careful
and add slowly as a dramatic eruption can occur if the temperatures are higher.
Step 16
Stir well using a stirring magnet at 3000rpm for duration of 10 minutes to ensure the

Page | 46
RM PHARMACEUTICALS
Page 4 of 4
ingredients are well mixed.
Step 17
Remove the beaker from heating/stirring mantle and leave to cool and remove the stirring
magnet from the beaker using a magnetic rod.
Step 18
Reheat the contents of the beaker, while reheating be sure to keep stirring using a glass rod
for 5minutes as bubbles may appear if the contents are not stirred.
Step 19
Weight out a plastic container on an analytical balance.
Weight of the container: 23.0664g
Step 20
Remove the beaker from the heating/stirring mantle and place the contents of the beaker into
the container and leave to cool before covering.
Theoretical yield Calculation
7.0616 (Beeswax) + 0.3802 (Borax) + 30 (Mineral Oil) + 20 (Water) 2.0023g (BDM) +
1.5043 (MBC) + 3.0058g (BZ3) = 63.9542g
Signed: _____________ (QC Manager) Date: _____________
Actual yield Calculation:
Container 23.0664g
Container & Formulation = 77.8043g
77.8043g – 23.0664g = 54.7379g

Page | 47
RM PHARMACEUTICALS
Page 1 of 4
Chemical Amount
Beeswax 8.91184g
Olive Oil 32ml
Butyl Methoxydibenzoylmethane (BDM) 4.5g
Octyl salicylate (OS) 2.5ml
Octyl-methoxycinnamate (OMC) 1.5g
Health and Safety:
2.0 Materials:
1.1 Reagents
 Beeswax
 Borax
 Distilled water
 Mineral oil
1.2 Equipment
 Magnetic rod

Page | 48
RM PHARMACEUTICALS
Page 2 of 4
 1ml pipette (x2)
2.0 Method
Step 1
Weight out accurately 8.91184g ± 0.1g of beeswax on an analytical balance.
Step 2
Step 3
Weight out accurately 4.5g ± 0.1g of Butyl Methoxydibenzoylmethane (BDM) on an
analytical balance.
Step 4
Accurately measure out accurately 2.5ml ± 0.1ml of Octyl salicylate (OS) using a 1ml glass
pipette.
Actual Volume: 2.5ml
Step 5
Accurately measure out accurately 2.5ml ± 0.1ml of Octyl-methoxycinnamate (OMC) using a
1ml glass pipette.
Step 6
Accurately measure out 30ml of olive oil using a 50ml graduated cylinder.

Page | 49
RM PHARMACEUTICALS
Page 3 of 4
Actual Volume: 30ml
Step 7
Actual Volume: 24ml
Step 8
Place the beeswax and the mineral oil into a 500ml glass beaker and place the beaker on the
Step 9
Step 10
Step 11
Step 12
Place the water into a different 100ml beaker and place the beaker on the heating/stirring
Step 13
Step 14
When the borax is dissolved fully add the BDM, OMC and OS to the beaker.
Step 15
Add the water/borax, BDM, OMC and OS into the beaker containing the oil/wax. Be careful
and add slowly as a dramatic eruption can occur if the temperatures are higher.
Step 16

Page | 50
RM PHARMACEUTICALS
Page 4 of 4
Step 17
Step 18
Step 19
Step 20
8.9147 (Beeswax) + 0.6297 (Borax) + 30 (Olive Oil) + 24 (Water) + 4.500g (BDM) + 2.5
(OMC) + 2.5 (OS) = 73.0444g
Container 23.1375g
Container & Formulation = 80.7009g
80.7009g – 23.1375g = 57.5634g

Page | 51
RM PHARMACEUTICALS
Page 1 of 4
Chemical Amount
Beeswax 8.7073g
Mineral Oil 18ml
Lanolin 13.9317g
Butyl Methoxydibenzoylmethane (BDM) 2g
Methylbenzildene camphor (MBC) 1.5g
Octocrylene (OCR) 2.5ml
Health and Safety:
3.0 Materials:
1.1 Reagents
 Beeswax
 Borax
 Distilled water
 Mineral oil
1.2 Equipment
 Magnetic rod

Page | 52
RM PHARMACEUTICALS
Page 2 of 4
 1ml pipette (x2)
2.0 Method
Step 1
Weight out accurately 8.7073gg ± 0.1g of beeswax on an analytical balance.
Step 2
Step 3
Weight out accurately 13.917g ± 0.1g of lanolin on an analytical balance.
Step 4
Weight out accurately 2g ± 0.1g of Butyl Methoxydibenzoylmethane (BDM) on an analytical
balance.
Step 5
Weight out acurately 1.5g ± 0.1g of Methylbenzildene camphor (MBC) on an analytical
balance.
Actual Volume: 1.500g
Step 6
Accurately measure out accurately 2.5ml ± 0.1ml of Octocrylene (OCR) using a 1ml glass
pipette.

Page | 53
RM PHARMACEUTICALS
Page 3 of 4
Step 7
Accurately measure out 18ml of mineral oil using a 50ml graduated cylinder.
Actual Volume: 18ml
Step 8
Actual Volume: 21ml
Step 9
Place the beeswax and the mineral oil into a 500ml glass beaker and place the beaker on a
Step 10
Step 11
Step 14
Step 15
Place the water into a different 100ml beaker and place the beaker on a heating/stirring
Step 16
Step 17
When the borax is dissolved fully add the BDM, MBC and OCR to the beaker.
Step 18
Add the water/borax, BDM, MBC and OCR into the beaker containing the oil/wax. Be
careful and add slowly as a dramatic eruption can occur if the temperatures are higher.

Page | 54
RM PHARMACEUTICALS
Page 4 of 4
Step 19
Step 20
Step 21
Step 22
Step 23
8.7890 (Beeswax) + 0.6164 (Borax) + 13.9865 (lanolin) + 18 (Mineral Oil) + 21 (Water) 2.0
(BDM) + 1.5 (MBC) + 2.5 (OCR) = 68.3919g
Container 23.1748g
Container & Formulation = 75.6845
75.6845g – 23.1748g = 52.5097g

Page | 55
4.0 Standard Operating
procedures (SOPs)

Page | 56
RM PHARMACEUTICALS LTD Page 1 of 3
SOP NO. QC120.410 Supersedes: N/A
Title: The Preparation of a 1% acetic acid solution.
Prepared by:__________ Date: 17/1/12
QC Analyst
Approved by:__________ Date: 17/1/12
QC Manager
Revision History: none
Issue: 1.0
Changes: none
1. Purpose:
To describe the procedure for preparing a 1% acetic acid solution.
2. Scope:
This SOP is applicable to all personnel carrying out quality control testing.
3. EHS Statement:
Refer to the relevant risks assessments.
4. Abbreviations:
Commonly used abbreviations in this SOP are documented in the respective sub sections.
Abbreviations
SOP Standard Operating Procedure
QC Quality Control
ml Millilitres
L Litre
HPLC High Performance Liquid Chromatography
N/A Not Applicable
R Instrument grade
5. RelatedDocuments
5.1 Impacted SOPs / Cross ReferencedSOPs

Page | 57
RM PHARMACEUTICALS LTD
Page 2 of 3
SOP NO. QC120.410 Supersedes N/A
Title: The preparation of a 1% acetic acid solution.
QC 120.340 Analysis of Product by High Performance Liquid Chromatography.
QC120.460 The preparation of 30:70 1% acetic acid: ethanol mobile phase for High
Performance Liquid Chromatography.
6. Responsibility:
6.1 It is the responsibility of the QC Specialist to revise this Sop and any associated
documents ensuring that they are an accurate up to date description of the current process.
The QC specialist also checks signs all completed testing to ensure that the tests are
completed and the paperwork is in order.
6.2 It is the responsibility of the QC Manager to approve QC SOPs and Specifications and
to assign the batch disposition on completion of testing.
6.3 It is the responsibility of the QA Manager to approve QC SOPs and Specifications
and to ensure that they are in accordance with relevant regulations, Quality Policies and
procedures.
7. Materials Required
100% acetic acid R
Ultra-Pure water
8. Equipment
1L volumetric flask
Reagent bottles
Disposable Plastic Pipettes
1ml pipette
9. Procedure:
Note. All standards and samples must be made up in mobile phase
Mobile Phase Preparation
70% methanol
30% water (distilled)
9.1 Wash a 1ml pipette with ultra-pure water.
9.2 Accurately measure out 3ml of 100% acetic acid R using a 1ml pipette into a 1L
volumetric flask.
9.3 Wash a 300ml graduated cylinder with ultra-pure water

Page | 58
Title: The preparation of a 1% acetic acid solution.
9.4 Accurately measure out 297ml of ultra-pure water using a 300ml graduated cylinder
into the same 1L volumetric flask.
9.5 Stopper and invert the flask a few times to ensure a uniform solution.
9.6 This produces a 1% acetic acid solution.
End of Document.

Page | 59
Title: The preparation of 30:70 1% acetic acid: ethanol mobile phase for High Performance
Liquid Chromatography.
Prepared by:__________ Date: 17/1/12
QC Analyst
Approved by:__________ Date: 17/1/12
QC Manager
Issue: 1.0
Changes: none
10. Purpose:
To describe the procedure for preparing 30:70 1% acetic acid: ethanol mobile phase for
HPLC analysis.
11. Scope:
12. EHS Statement:
13. Abbreviations:
Abbreviations
QC Quality Control
ml Millilitres
L Litre
µm Micrometre
N/A Not Applicable
R Instrument grade

Page | 60
Title: The preparation of 30:70 1% acetic acid: ethanol mobile phase for High
14. RelatedDocuments
QC 120.430 Operation of an Agilent 1050 series HPLC instrument.
QC 120.470 The Preparation of a 1% acetic acid solution.
15. Responsibility:
procedures.
Ethanol R
100% acetic acid
Distilled water
17. Equipment
Mobile Phase Filtration Apparatus
500ml graduated cylinder
1L volumetric flask
500ml Reagent bottles x2
18. Procedure:
Mobile Phase Preparation
30% 1% acetic acid
70% Ethanol R

Page | 61
Title: The preparation of 30:70 1% acetic acid: ethanol mobile phase for High
18.1 Wash a 1 litre volumetric Flask with distilled water and wash
250ml graduated cylinder distilled water.
18.2 Accurately measure out 300ml of 1% acetic acid using a 500ml
graduated cylinder and place it into the 1L volumetric flask.
18.3 Accurately measure of 700ml of ethanol R using a clean 500ml
graduated cylinder and place it into the 1L volumetric flask.
18.4 Set up the HPLC filtration apparatus and filter the mobile phase
through a 0.45µm filter paper.
18.5 When the mobile phase is completely filtered, pour the mobile
phase into two 500ml reagent bottles.
18.6 This is now a 30:70 1% acetic acid: ethanol mobile phase.
End of Document.

Page | 62
Title: Operation of an Agilent 1050 series HPLC instrument.
Prepared by:__________ Date: 24/1/12
QC Analyst
Approved by:__________ Date: 24/1/12
QC Manager
Issue: 1.0
Changes: none
19. Purpose:
To prepare a procedure for the operation of an Agilent 1050 series HPLC instrument.
20. Scope:
21. EHS Statement:
22. Abbreviations:
Abbreviations
QC Quality Control
ml Millilitres
L Litre
N/A Not Applicable
R Instrument grade

Page | 63
23. RelatedDocuments
QC 120.470 The Preparation of a 1% acetic acid solution.
QC 120.420 The preparation of 30:70 1% acetic acid: ethanol mobile phase for High
24. Responsibility:
procedures.
Ethanol R
1% acetic acid
26. Equipment
Glass vials
Plastic pipettes
27. Procedure:
Note. It is the responsibility of the analyst to follow this SOP step by step and it is the
responsibility of the laboratory supervisor to train the analyst on how to operate the
instrument properly.
27.1 On the computer desktop click on instrument 1 online.
27.2 Open the purge valve click on instrument set up pump set flow rate to
5ml/min click on instrument more pump controlclick on for the pump.

Page | 64
27.3 Purge column until all the air bubbles are removed. Click on instrument set up
pump set flow rate back to 1ml/min and close the purge valve.
27.4 Click on method.
27.5 Click on edit entire method.
27.6 Set flow rate to 1.000ml/min.
27.7 In the solvents sub section A enter 30:70 and in description enter 1% acetic acid:
Ethanol.
27.8 Set stop time to 10.00mins.
27.9 Set Posttime to off.
27.10 Click ok.
27.11 Injection section pops up. Set the following parameters
Injection volume: 10µl
Draw speed: 200µl/min
Eject speed: 200µl/min
Draw position: 0.0min
27.12 Click Ok.
27.13 Set detector wavelength to 313nm.
27.14 Click ok.
27.15 Signal details.
27.16 Click ok.
27.17 Save method as e.g. filters and give subdirectory.
27.18 Click on Sequence.
27.19 Click onto sequence parameters select sequence output printer file.
27.20 Go into Sequence table.

Page | 65
27.21 Set Sequence Table e.g. BDM, Injection 1, method filters and click ok.
27.22 Save sequence as e.g. UV filters.
27.23 Click on Instrument and go into More Pump  Control  click on for the Pump.
27.24 Click on Instrument again and go into More VWD  Control  click on for the
light.
27.25 Go to Run Control.
27.26 Ensure all critical windows are green.
27.27 Click Run Sequence.
End of Document.

Page | 66
Title: The preparation of BDM, BP3 and MBC stock solution and standards.
Prepared by:__________ Date: 24/1/12
QC Analyst
Approved by:__________ Date: 24/1/12
QC Manager
Issue: 1.0
Changes: none
1. Purpose:
To describe the procedure for preparation of BDM, BP3 and MBC stock solution and
standards.
2. Scope:
3. EHS Statement:
4. Abbreviations:
Abbreviations
µL Microliter
QC Quality Control
ml Millilitres
N/A Not Applicable
ppm Parts per million
Min Minute
R Instrument grade
BDM Butyl Methoxydibenzoylmethane

Page | 67
MBC 4-Methylbenzylidene
BP3 Benzophenone
5. RelatedDocuments
QC120.450 The preparation of BDM, OMC and OS stock solution and standards.
QC120.460 The preparation of BDM, MBC and OCRstock solution and standards.
6. Responsibility:
procedures.
BDM
MBC
BP3
Ethanol R
8. Equipment
100ml volumetric flasks (x3)
10ml volumetric flasks (x15)
Plastic pipette
Funnel
Tin foil
25ml glass beakers (x3)
Sonicator
9. Procedure:
A: Preparation of BDM, BP3 and MBC stock solution.

Page | 68
9.1 Weight out 100mg of BDM, MBC and BP3 in separate 25ml glass beakers on an
analytical weighting balance.
9.2 Add 10 millilitres of ethanol to each of the three beakers using a 10ml graduated
cylinder and transfer them into a sonicator for 15 mins to mix the solution.
9.3 Place a weight on the top of each beaker to ensure they do not tip over while in the
sonicator.
9.4 Transfer the solutions from the beakers into three clean 100ml volumetric flasks.
9.5 Fill the volumetric flasks up to the mark with ethanol using a funnel.
9.6 Place a stopper on the flasks and invert a few times to ensure a uniform solution.
These are now 1000ppm stock solutions of three UV filters.
B: Preparation of BDM, BP3 and MBC Standards Ranging from 100ppm to 500ppm.
9.7 Accurately measure 1ml of the 1000ppm BDM stock solution using a 1ml pipette.
9.8 Transfer into a 10 ml volumetric flask and make up to the mark with ethanol R. This
is now a 100ppm standard.
9.9 Repeat this step using 2ml, 3ml, 4ml and 5ml of the BDM stock solution to make the
200ppm, 300ppm, 400ppm and 500ppm standards respectively.
9.10 Accurately measure 1ml of the 1000ppm BP3 stock solution
using a 1ml pipette.
9.11 Transfer into a 10 ml volumetric flask and make up to the mark
with ethanol R. This is now a 100ppm standard.
9.12 Repeat this step using 2ml, 3ml, 4ml and 5ml of the BP3 stock
solution to make the 200ppm, 300ppm, 400ppm and 500ppm standards.
9.13 Repeat steps 9.7-9.9 for the MBC standards.
End of document.

Page | 69
Title: The preparation of BDM, MBC and OCR stock solution and standards.
Prepared by:__________ Date: 6/3/12
QC Analyst
Approved by:__________ Date: 6/3/12
QC Manager
Issue: 1.0
Changes: none
10. Purpose:
To describe the procedure for preparation of BDM, OMC and OS stock solution and
standards.
11. Scope:
12. EHS Statement:
13. Abbreviations:
Abbreviations
µL Microliter
QC Quality Control
ml Millilitres
N/A Not Applicable
ppm Parts per million
Min Minute
R Instrument grade
BDM Butyl Methoxydibenzoylmethane

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