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Aloe Vera is known as a TRUE ALOE. It is largely used in herbal medical specialties so it is besides known as FIRST AID PLANT or MEDICINAL PLANT. The ALOE word was derived from the aerophilic word-‘alloeh ‘ which means “ Acrimonious SUSSTANCES ” . Aloe is an demulcent rosin with the softening belongingss. It belongs to household Liliaceae [ 1 ] .

1.1.1 History: –

Aloe vera was considered to one of best medicative works ot used in the intervention of assorted wellness status. It was foremost understood by Egyptians to holding multiple nature of it belongingss [ 19 ] . But Aloe Vera was foremost discovered in 1862 by German Egyptologist -George Fbers. But its first English interlingual rendition was found in 1655 by John Goodyear in Dioscorider ‘ de materia medica, and he wrote aloe is really better in gustatory sensation.

Aloe Vera has been used by many different civilizations but Indians, Chinese, Greeks, Romans, have all used aloe Vera as a medicinal or healing works. Botanists have find more than 300 species of aloe Vera like Rumex, barbadensis etc. Out of this species, merely five have strongest medical benefits likes Aloe barbadensis Miller, Aloe perryi baker, Aloe ferox, Aloe Saponaria, Aloe arborescens. Aloe barbadensis Miller is largely widely used and most powerful every bit good. Throughout the history of aloe vera, the works is in assorted signifier holding tropical properties-refered as ‘plant of immorality ‘ . It is belonging to lily household because flower green goods yearly in the spring which resembles of ester lily. [ 1-2 ] .

1.1.2 Common Name: –

Chinese aloe, True aloe, Indian aloe, First assistance works, Medicinal works, Miracle works, Barbadose aloe, Burn aloe and many more [ 1 ] .

1.1.3 SCIENTIFIC Name: –

Aloe barbadensis, Aloe capensis [ 1 ] .

1.1.4 Alternate Name: –

Aloe Vera has a different name in worldwide like, In India-Ghrtakumari But in Gujarat and Rajasthan it ‘s known as Gwarpatha, In Pakistan-quargandal which is used in unani medical specialty, In Indonesia-lidah buaya, In Thailand- crocodile tail works, In south Africa-sabila [ 2 ] .

1.1.5 Description: –

Aloe Vera grows in to the drab climes or land. So it is largely found in the India, Africa, Caribbean, and other dry climes.

Aloe Vera is a short root or stems less works. It chiefly contains foliages and flowers. Aloe foliages are in green in coloring material and midst with the same assortment. The size of foliages is 50-60 centimeter long and 4-5 centimeter midst. The border of the foliages is serrated. But workss bear flowers one time in a twelvemonth in summer season. Flowers contain cannular neckband which is xanthous in coloring material and contain ‘aloe tic ‘ juice can be collected by cutting the foliages near to root. The construction of aloe foliage shows an outer cerebral mantle which is difficult couple to the nowadays of Ca and Mg. Tubes of xylem and bast were found below the cerebral mantle and its supplied H2O and minerals to foliages [ 1 ] .

1.2 CHEMICAL CONSTITUTE: –

Aloe Vera has weird medicative belongingss. Botanists have found more than 200 of import nutritionary components in aloe Vera foliage which holding to execute a map. They are MINERALS like Calcium, Sodium, Copper, Zinc, Iron, and Manganese ( Essentials for castanetss, Regulates acidic or alkaline degree of organic structure fluid ) , VITAMINS like Vitamin A, Vitamin C, Vitamin E, Vitamin B12, and Folic Acid ( To develop new blood cells ) , ENZYMES like lipase, Peroxidise ( Helps dyspepsia ) , SUGAR like Monosaccharide ‘s and polyoses include Sucrose, lactose ( Maintain cholesterin degree, Improve the strengthening of castanetss ) , LIGNINS which are the Cellular substances which has n’t medicative benefits But it has an accessing belongings. AMINO ACID which are required by human organic structure provided by aloe Vera like Leucine, Isoleucine, Valine, Theonine, Lysine, Methionine, Phenylamine, STEROL like Camp steroid alcohol, sitosterol ( Pain killing belongingss ) , ANTHRAQUINONES like Aloin, Anthranol, Aloetic acid, Barbaloin, Isobarbaloin and Aloe emodin

Aloe emodine [ 4 ] Aloin [ 3 ]

And OTHERS like Salicylic acid, Tannins, Monosulfonic acid, Acemannam, Water [ 1-2 ] .

Aloes Vera chiefly contain 96-99 % of H2O and constitutes are in rare portion. So it ‘s work in based on “ SYNERGISTIC ACTIONS “ which means ‘working together of two or more substance and give greater action as comparison to individual substance. This phenomenon was explained by Dr.Atherton [ 1-2 ] .

1.3 CULTIVATION OF ALOE VERA [ 1-5 ] : –

Aloe Vera grown commercially for its high demand in decorative industries and intervention of mending in India. It has medicative belongingss due to its acrimonious gustatory sensation. Aloe Vera grown in a rainless climates or land. It ‘s ca n’t remain in cold temperature. So it is grown in all portion of South Africa and India like Rajasthan, Gujarat, Maharashtra and many more.

Soil demand for all species are harsh flaxen loam dirt with some birthrate. In add-on, it is noticed that its growing was easier and faster in black cotton dirt in cardinal India with the about by acidic pH.

For the perfect growing of Aloe Vera, lane should non administer below the degree of 20-30cm. Aloe Vera need a level land and should be exact degree with 2-3 agriculture. Plant should works with some distance because of irrigation. Distance between two Fieldss was 10m Ten 3m.

The proper clip of chumps should be planted in month of July-August in monsoon season because chumps get proper H2O to turn up and endurance. Chumps should hold 3-4 oral cavity old holding 3-4 foliages and 20-22 degree Celsius long. About 30,000 chumps needed for one hectare planting. After seting chumps, dirt around the root should press and blow must be made proper to avoid the lethargy.

Irrigation is necessary a critical phase to proper growing of works. First irrigation is required after seting chumps followed by 4-6 per twelvemonth which is cut downing by every clip. Weeding should be carried out after seting the works and two or three weeding carried out throughout twelvemonth.

All the species of Aloe Vera are unsusceptible to most of insects and plagues from any portion of county. But sometime bugs, leaves points have been reported in some portion of state because of light irrigation.

By and large yield obtained from 2 to 5 twelvemonth after plating. Average output after 2 twelvemonth is around 15-20 t/ha fresh foliages. Well managed irrigated harvest give around 30-35 t/ha fresh foliages. Fully good developed foliages give an ‘ALOE JUICE ‘ .

After reaping, selling of Aloe Vera leaves is non to the full developed in our state.

1.4 Use: –

Aloe Vera is good known for its medicinal belongingss since centuries. The works is able to bring around a broad scope of disease. The portion of works like foliages, foliage gel is used clinically. Some several utilizations of Aloe Vera are ( 1 ) EXTERNAL USES-Use in intervention of Burnss, allergic reaction, lesions, acne, Rheumatoid arthritis, Rheumatic febrility, Acid dyspepsia cuts, Inflammatory status of digestive system, Sun burn ulcers, Kill mould, bacterium, viruses, fungus, Wrinkles etc ( 2 ) COMMERCIAL Use: – Aloe Vera is widely usage in cosmetics and many more like in unctions, pills, jelly, lotion, bevareges.as a grocery etc [ 1-6 ] .

( 3 ) MEDICINAL USE-They are Treatment of diabetes because it cut down blood sugar, Treatment of AIDS, Protect from lung malignant neoplastic disease, Aloe juice is used for ingestion issues, Prevent fungous infection, Prevent vaginal infection, Prevent scarring, Eczema, Constipation, Intestinal infection, Relieves from candiala, Treatment of hyperglycemia, Maintain degree of cholesterin, Injection of Aloe Vera extracts to handle malignant neoplastic disease, Skin upset. And some of general usage like Antibacterial belongingss, Antifungal belongingss, and Prevent radiation induced hurts, Inhibit growing of streptococci species in vitro [ 1-6 ] .

1.4.1 ALOE Preparation: –

some of the aloe readying which are widely use like aloe vera shampoo, aloe vera lotion, aloe vera gel, aloe vera juice, aloe vera feeding, aloe vera butter, aloe vera dried herbs pulverizations, aloe vera shower gel, and aloe vera vegetarian capsules [ 1 ] .

1.4.2 SIDE EFFECT AND SAFETY PRECAUTION: –

Aloe Vera has a medicative or mending belongingss, it does come with some disadvantage. Aloe Vera gel, aloe Vera pick does non holding major side consequence but other readying like juice, shampoo butter, holding certain hindrance like, Diarrhea, Blood electrolyte instability, Constipation, Muscle failing, Abnormal bosom beat [ 7 ] . Aloe Vera injection is to be avoided because of holding human deaths. It can do decease with malignant neoplastic disease patients Aloe Vera should be avoided while gestation, kids, chest eating [ 7 ] .

ALPHA-GLUCOSIDES INHIBITORS: –

1.5 INTRODUCTION TO ALPHA-GLUCOSIDE ENZYME: –

It is a category of a medicine for type-2 diabetes which decrease blood sugar degree by diminishing saccharides from the enteric. Discovery of alpha-glucosides inhibitors has been really utile to develop curative for the intervention for the carbohydrate- mediated disease like diabetes. Two categories of drugs like glycosidase inhibitors and lipase inhibitors which lower blood glucose by altering the soaking up degree of fat and carbohydrates [ 8-9 ] . Alpha – glucosides inhibitors like acarbose, miglitol, voglibose have been surveies in Europe state but some of these are besides available in united province [ 8 ] . Alpha -glucosidase inhibitors cut down the impact of saccharides blood sugar by inhibit the upper degree of GI [ 8 ] .

1.5.1 MECHANISM OF ACTION: –

Alpha glucosides inhibitors are competitory inhibitors of 1alpha glucosidase which are located in the coppice boundary line of little enteric ( epithelial cells ) . These inhibitors bind to the oligosaccharide adhering site of the enzyme and hold digestion of polysaccharide ensuing slow down nutrient digestion in intestine [ 10 ] .

While the GI piece of land dosage non play a of import function in the remedy of diabetes, but altering its physiological activity can be used to command disease. In these instance alpha-glucosides inhibitors are used. Alpha glucosides delay digestion of saccharides by hydrolyses of oligosaccharides into monosaccharose. Alpha glucosidase inhibitors can be used to cut down glycemic excussions and hypoglycemia holding type-1 diabetes. Furthermore it is used in the intervention of patients with type-2 diabetes and its lessening plasma triglyceride [ 11 ] .

Alpha glucosides inhibitors have grounds helpful for the people with diabetes who have n’t able to maintain their blood sugar degree within a safe scope. In such instance inhibitors like acarbose and miglitol aid to maintain the blood sugar degree in safe scope by decelerating a rate of enteric which absorbed sugar from blood while eating. These inhibitors can do low blood sugar while used in combination with other medicine for diabetes or with insulin. Diabetic people who are on a regular basis utilizing insulin but once they are use alpha glucosidase inhibitors than they cut down usage of insulin [ 12 ] .

CHAPTER- 2

High PERFORMANCE LIQUID CHROMATOGRAPHY.

2.1 Introduction: –

Chromatography is the separation of a mixture into individual constituents by utilizing nomadic and stationary stage. High public presentation liquid chromatography is widely usage in analytical chemical science and industrial degree to place constituents. It is one of the fastest turning techniques in pharmaceutical industry for analysis of mixture os substance. It is besides knows every bit high force per unit area liquid chromatography. It is a high improved as comparison to column chromatography. Smaller atom size tins ( 3-20Aµ ) be analysed by hplc and allows much better separation of a constituents. Detection method is used in hplc which are extremely automated and highly sensitive as comparison to column chromatography [ 13-14 ] .

Some of the advantage over column chromatography is, ( a ) little stationary stage are used with widely scope available, ( B ) column which are used in hplc which are made up of metal and little in size so no fright to breakage, ( degree Celsius ) hplc is available in analytical and preparatory graduated table. But some of disadvantage over column chromatography like cost of equipment is high so handle with attention, runing force per unit area is high ( 500-3000 pounds per square inch ) [ 14 ] .

Basic rule is base on surface assimilation. When a mixture of constituents is introduced in to column, they are travel harmonizing to their affinity. The constituents which has a more affinity towards adsorbent, travel slower and frailty versa [ 14 ] .

A®NORMAL PHASE HPLC: –

Here, stationary stage is polar in nature and nomadic stage is non polar in nature. In this method non polar constituents eluted foremost because of less affinity. The column is fitted with a silicon oxide gel and hexane used as a dissolver. Column length is 150-250 millimeter and less than 4.6 mm diameter [ 13-14 ] .

A°REVERSE PHASE HPLC: –

Here, stationary stage is non polar and nomadic stage is polar in nature. Polar constituents eluted foremost. Column size is same but modify to do a non polar by utilizing long concatenation of hydrocarbons like C8, C12, C4, octadecyl and mixture of H2O and intoxicant usage as a dissolver. In this instance strong attractive force between polar dissolver and molecular in the mixture passed through column. So it is common stage of hplc [ 13-14 ] .

2.2TYPES OF HPLC TECHANIQES: -There are major four type of hplc as below,

( 1 ) Partition chromatography: –

In 1952, Archer John Porter Martin and Richard Laurence Millington Synge were awarded a Baronial award in chemical science to development of these techniques. So it is first techniques which developed for the separation of constituents like amino acid. These chromatography rules are besides applied to the thin bed chromatography, paper chromatography to divide the constituents.

( 2 ) Adsorption chromatography: –

The chief hplc rule is based on surface assimilation. Here constituents can be separated because of difference in affinity of constituents towards normal or rearward stages [ 14 ] .

( 3 ) Ion exchange chromatography: –

It is most often used chromatography for the separation and purification of protein, nucleic acid, polypeptides, and other charged molecular. It is a succeful technique because of its high capacity, simpleness, and high declaration power. The chief rule is base on reversible exchange of ions between ion nowadays in the solution and those present in ion exchange rosin [ 15 ] .

A®Cation exchange: –

Solid-H+ + M+ === solid-M+ + H+

( Solution ) ( Solution )

A®Anion exchange: – Solid-OH- + A- ==== solid-A- + OH-

( Solution ) ( Solution ) . [ 14 ]

Separation in ion exchange chromatography depends upon reversible surface assimilation of charged solute molecular to ion exchange group of opposite charge. Some of the functional group used in ion exchange chromatography is [ 16 ] ,

Name OF ANION

FUNCTION GROUP

Diethyl amino ethyl

-O -CH2-CH2-N+H ( CH2CH3 ) 2

Quatemary amino ethyl

-O-CH2-CH2-N+H ( C2H5 ) 2-CH2CHCH-CH3

Name OF CATION

FUNCTION GROUP

Carboxymethyl

-O-CH2-COO-

SULPHOPROXY

-O-CH2-CHOH-CH2-O-CH2-CH2SO3-

( 4 ) Size exclusion chromatography: –

It is besides know as a gel chromatography. In 1959, Porath and Flodin described the separation of H2O soluble constituents by this chromatography. Equally shortly as gel had commercially available, they were extensively used for the dividing purpose [ 17 ] . Here gel used as molecular screen and hence mixture of substance with different molecular sieze are separated. Soft gel like dextran, polyacrylamide are used [ 14 ] .

It is used to analysis of man-made polymers and oligomes, lipoids, natural macromolecular like protein, glucose, cellulose derived functions, and rough oil olefines [ 17 ] .

HPLC techniques besides has two different category like ( a ) analytical HPLC-it is used when analysis really little sum of sample are needed. And ( B ) preparatory HPLC- it is used when separation of few gm of mixture by HPLC. But it is besides can sort on base of separation and there are ( a ) analytical HPLC: – Where merely analysis of the samples is done. Recovery of the samples for reusing is usually non done, since the sample used is low. Eg. milligram measures. And other is ( B ) preparatory HPLC: – Where the single fractions of pure compounds can be collected utilizing fraction aggregator. The gathered samples are reused eg. Separation of few gms of mixtures by HPLC [ 14 ] .

2.3 INSTRUMENTATION [ 13-14 ] : –

Brief debut of instrumental techniques are as follows,

( 1 ) Pump: –

Mechanical and pneumatic pump are used in hplc. Mechanical pump operates with changeless flow rate where pneumatic pump operates with changeless force per unit area. It ‘s necessary to utilize pump because dissolver must passed through column at high force per unit area.

( 2 ) SOLVENT DEGASSING: –

In isocratic techniques, nomadic stage are prepared by utilizing mixture of dissolvers, than passed through column with under high force per unit area ensuing bubble formed, so it can non give proper consequence or steady baseline. So before utilizing solvent mixture in HPLC, degassing is necessary. So some of degassing technique is use like ultrasonication.

HPLC

( 3 ) Column: -Two types of column used in HPLC,

A® Guard column: –

It is besides known as safety column. Any impurities nowadays in dissolver or nomadic stage which can foretell by guard column.

A®Analytical column: –

It widely use which can decided efficiency of separation. Different column size is available depending upon separation. Column made up of chromium steel stell, glass, poly ether ether kiton etc.

( 4 ) Detector: -They are

( a ) Ultraviolet sensors: –

Many organic compounds captive UV visible radiation of different wavelength. Two type of UV sensors are available depends on wavelength- short wavelength and long wavelength. By UV sensor we can acquire direct reading is how much visible radiation is observed.

( B ) Flurometric sensors: – It can be used for the substance which can absorbed blossoming visible radiation.

( degree Celsius ) Conduction sensors

( vitamin D ) Refractive sensors

( vitamin E ) PDA sensors which are similar to UV sensors.

( 5 ) Recorder: –

They are used to recorded response from sensors. It can enter base line and keeping clip of extremum. Now a twenty-four hours computing machine is used to entering informations.

2.4 HPLC PARAMETERS: –

2.4.1 INTERNAL Diameter: – Internal diameter of the hplc column play an of import function in sensing or separation of compounds. For the research lab usage little column was used but in industrial degree large column was used.

2.4.2 RETENTION Time: – it is a defined as a clip between point of injection and visual aspect of extremum. It step in minute or seconds.

2.4.3 RETENTION VOLUME: – Retention volume in the volume of nomadic stage required eluting 50 % of the compounds from the column,

Retention clip = Retention time.Flow rate.

2.4.4 PUMP Pressure: – Pumps which are used in hplc really in force per unit area depends on separation. Modern HPLC system has been improved to work at high force per unit area so they are able to divide little atom size from mixture.

2.4.5 Theoretical Home plate: – It is a functional unit of the column. Distribution of solute between stationary stage and nomadic stage has attained equilibrium in the home base. High of the home base depends on efficiency of separation. It is knows as a HETP ( HIGHT EQUIVELENT TO THEORITICAL PLATE ) . If HETP is less, than column is more efficient and if HETP is more, than column is less efficient.

HETP is given by Van Deemter equation

HETP = A + ( B/u ) + Cu

Where,

A = Eddy diffusion term or multiple way diffusion which arises due to packing of the column. This is unaffected by nomadic stage speed or flow rate. This can be minimised by uniformity in wadding.

B = Longitudinal diffusion term or molecular diffusion which depends on flow rate.

C = Effect of mass transportation which depends on flow rate.

u = Flow rate or speed of the nomadic stage.

2.5 APPLICATION OF HPLC: –

High public presentation liquid chromatography has a big divergency of application. Initially it was merely used to analysed antibiotic, vitamins by development of contrary stage [ 18 ] .

High public presentation liquid chromatography can analyze really minor sum of substance like monograms at analytical graduated table to micrograms at preparatory graduated table. It is used in a nutrient analysis, biotechnology, biochemical separation, pharmaceutical field, and chemical industries like dyes, fatty acid, toilet articles merchandises [ 14 ] .High public presentation liquid chromatography has a easy and good duplicability so it has been widely usage in clinical research lab. One of the initially application of hplc in the field of clinical was quantification of theophyline in wheezing baby [ 18 ] .

Some of of import applications are as likes, ( a ) Isolation of natural merchandise, ( B ) Quantitative analysis or check of merchandise, ( degree Celsius ) Identify, analyzing and purification of compound mixture, ( vitamin D ) To look into pureness of compound mixture, ( vitamin E ) To happen physical belongingss of compounds, ( degree Fahrenheit ) Used in analyzing H2O pollution [ 18 ] .

The measure of sum is besides determined by hplc which including following method [ 14 ] ,

A® By comparing criterion and sample peak country, the measure of sample can be determined which is known as a DIRECT COMPRISION METHOD.

A® To happen concentration of sample by plotting graph between peak country vs. concentration of the standard drug and intrapolation of unknown sample which is known as CALIBRATION METHOD.

Hplc play an of import function in drug find with the combination of NMR. It is besides utile to place assorted chemical species with the combination with mass spectrometry. It play valuable function in curative drug monitoring to separation of drug from plasma which is speedy procedure [ 14 ] .

It has some advantages over other chromatography like, ( a ) Thin bed chromatography is cheap but it ca n’t give batter declaration to compare HPLC. ( B ) Mass spectrometry required a big capacity of maintain budget and skillfull individual to run chromatography but in HPLC it ‘s non high budgt. ( degree Celsius ) For the HPLC we have big figure of nomadic stage is available so we have versatility in pick of nomadic stage as comparison to other chromatography. ( vitamin D ) Atomatic injection system are available in HPLC, so its clip economy and give high declaration as comparison to other [ 20 ] .

2.6 LIMITATION OF HPLC: –

It is a clip devouring method. Analysis of the compound mixture, nomadic stage ( mixture of constituents ) are used ensuing big sum of waste solution are obtained [ 14 ] .

CHAPTER- 3

EXTERIMENT

3.1 Purpose: –

The purpose of undertaking was to cognize batter separation of standard solution of Aloe Vera root in nomadic stage by utilizing isocratic hplc techniques and happen enzyme activity on it by utilizing assay whether it is more powerful or less powerful.

3.2 CHROMATOGRAPHY Equipments: –

3.2.1 HPLC SYSTEM: –

This system is manufactured by Agilent engineering 1200 series with theoretical account no G1310A and consecutive no DE62956545.

3.2.2 SOFTWEAR USED: –

The package used was Microsoft Windows XP.

3.2.3 COLUMN USED: –

The column used was Kromaril 5C18 which is made up of chromium steel still.

3.2.4 APPARATUS USED: –

Pipette with different size available like 1000land 5000Aµl, Analytical balance, Volumetric flask ( 10ml, 20ml ) , Funnel, 100ml Beaker, Measuring cylinder ( 500ml, 50ml, 20ml,10ml ) , Filter paper which made by Fischer scientific with size 0.45Aµm, Filter holder made up of Pyrex trade name glass.

3.2.5 PREPARATION OF MOBILE PHASE: –

The nomadic stage used was a mixture of a methyl alcohol and H2O with ratio of 50:50. So take methyl alcohol and H2O, mix good, and filter to take any solid atom followed by degassing to take air bubble. Than it is ready for experiment. We are utilizing isocratic system so nomadic stage may change in concentration like, 20:80, 30:70.

3.2.6 PREPARATION OF STANDARD SOLUTION OF ALOE VERA: –

The standard solution of aloe vera root was provided by Dr.solomon sir and after that I have to thin in methyl alcohol to do up volume to 0.40mg/2ml.

3.3PROCEDURE: –

First set up the hplc system and run with nomadic stage for half an hours. Than inject standard solution of aloe Vera root and run for half an hours, take the graph. Now change the concentration of nomadic stage and once more repeat same process until you get batter separation. Then compare all graphs with each other and make to decision. Here we were utilizing two different wavelengths like 280nm and 360nm.

3.5 Assay: –

Assay technique was used to find enzyme activity of a substance. Here we were utilizing alpha-glucosidase enzyme. Basic rule involved in check was, phosphate buffer contain free group which has no coloring material but when it come across with standard solution, retain its coloring material after incubation. So color alteration like colourless to yellow coloring materials took topographic point.

Solution USED IN ASSAY: –

Here we have to fix solution for the check was 25Aµl, 1.2 U/ml alpha-glucosidase, 25 Aµl of phosphate buffer, standard solution of aloe vera, 25 Aµl of 2.5 millimeters pNPG and eventually if proper coloring material alteration was non occur than add ending solution 100 Aµl of 0.2 M Na2Co3.

3.5.1 PROCUDURE: –

1

2

3

4

Tocopherol

1

2

3

4

5

6

BLANK

Concentration in natural at no 1-0.50mg/ml, natural no 2- 0.25mg/ml, natural no 3- o.125mg/ml, and natural no 4- 0.0625mg/ml.

Here all the column from 1 to 6 filled by 25Aµl,1.2 U/ml alpha-glucosidase, so add 25Aµl of phosphate buffer, add 25Aµl of trial solution, and eventually add 25Aµl of 2.5 millimeter pNPG. And so reassign 2.5Aµl solution from low concentration to high concentration. Than kept mixture for some clip and incubate home base at 37A°C for 10 min. and tested optical density.

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