Selasa, 30 September 2014

TEORI DASAR SPEKTROFOTOMETRI UV-VIS (diambil dari webnya Shimadzu)

What Is Light

The question of whether light is wave or particle, throughout history, been the subject of much debate. It is well known that various theories developed from Newton's assertion that light is particle and Huygen's assertion that light is wave. In modern physics, it is now known that; while light has the wave nature, it also has particle-like properties. In this article, we will describe the dual nature of light and the relationship between matter and light absorbance.

1. The Dual Nature of Light

(1) Wave-Like Properties of Light

Fig.1 Electric Field and Magnetic Field in Light/Fig.2 Young's Experiment
Although light is generally said to be a wave, unlike the waves that occur at the surface of a body of water, it does not require a medium. As shown in Fig.1, light consists of an electric field and a magnetic field that intersect each other at a right angle as they move through a vacuum. The distance between successive peaks of either the electric field or the magnetic field is the wavelength.

When handling light, one encounters phenomena that are particular to waves, such as interference and diffraction. The experiment in which Young discovered the interference of light and concluded that light was a waveform is well known. As shown in Fig.2, the monochromatic light emitted from a light source, L, passes through a single slit, and then passes through two more slits, S1 and S2. As a result, interference fringes are observed on the screen at the back as a pattern of alternating strips of brightness and darkness. This can be explained by thinking of S1 and S2 as light sources in phase with each other. Waves travel from these light sources to the screen at the back. At points where the waves are in phase, they reinforce each other, whereas at points where the waves are out of phase, they cancel each other out. If one thinks of the surface of this paper as the surface of a body of water, and the slits as partitions with holes in them, then waves moving from left to right would behave in the same way. In this sense, Young's experiment demonstrates the wave-like nature of light in an intuitive way. Incidentally, the diffraction grating used in a UV-VIS spectrophotometer creates monochromatic light using the wave nature that light diffracts and causes interference.

Light consists of certain types of electromagnetic waves.
Electromagnetic waves are referred to by different names in accordance with their wavelength, as shown in Fig.3. "Light" usually refers to electromagnetic waves in the range spanning infrared radiation and ultraviolet radiation, but in some cases it refers only to visible light. Light with wavelengths in a range of approximately 400 to 800 nm is referred to as "visible light", and is the light that we humans can see with the naked eye.
For example, light with a wavelength of 470 nm is blue, light with a wavelength of 540 nm is green, and light with a wavelength of 650 nm is red. Visible light could be described as the kind of light that we humans are familiar with because of our ability to actually see it.
Fig.3 Classification of Light According to Wavelength
Fig.3 Classification of Light According to Wavelength

(2) Particle-Like Properties of Light

Next, let us look at the particle-like behavior of light. Among the developments that helped identify this behavior was a series of experiments on the photoelectric effect that were conducted in the late 19th century and the early 20th century.
The results of these experiments could not be explained by considering light as a wave, but they could be explained by considering it as a particle. When emphasizing the particle-like aspects of light, the term "photon" is used.
Fig.4 Concept of Photoelectric Effect
Fig.4 Concept of Photoelectric Effect3)
Fig.4 illustrates the basic concept of the photoelectric effect, a phenomenon in which electrons are emitted from a metal surface when light strikes it. The emitted electrons are called "photoelectrons". Even though no electrons are emitted when intense light strikes the surface, electrons are emitted when light of a shorter wavelength strikes the surface. If the wavelength of the light striking the surface is decreased, the number of electrons emitted does not change, but the energy of the electrons increases. If the light striking the surface is intensified, the number of emitted electrons increases, but the energy of the electrons stays the same. These phenomena cannot be explained by considering light as a wave, but they can be explained by considering it as a particle, with electrons being knocked out by these particles when they strike the metal. In combination with results of experiments on the Compton effect and other experiments, the particle-like properties were recognized. Incidentally, the photomultiplier tube that is used as the detector in UV-VIS spectrophotometers detects light using the photoelectric effect.

We have looked at the dual nature of light, with its mixture of wave-like and particle-like properties. The fact that light has these two opposing characteristics may seem strange, but this is the way light is modeled in modern physics.

2. Absorption of Light by Matter

A wide variety of information about a substance can be obtained by irradiating it with light. With a UV-VIS spectrophotometer, irradiating a substance with ultraviolet and visible light makes it possible to obtain information about the electrons in that substance, and it is even possible to measure its quantity.

Let us consider the absorption of light by matter. This is closely related to quantum mechanics. The theory of quantum mechanics was developed in the early 20th century, and forms part of the basis of modern physics. Quantum mechanics can be easily understood by comparing it to Newtonian mechanics.
Broadly speaking, Newtonian mechanics is a theory that relates to the motion of large particles, whereas quantum
mechanics is a theory that relates to the motion of small particles (e.g., atoms and molecules). Newtonian mechanics handles the motion of particles as a continuous entity, whereas quantum mechanics asserts that small particles exist in discrete states of motion (energies). At the time when Newtonian mechanics was the dominant theory, the concept of quantum mechanics was difficult for people to accept. Over time, however, its validity was demonstrated.
Fig.5 Energy Levels
Fig.5 Energy Levels
Solving the equations of quantum mechanics that relate to the electrons in an atom gives a model, like that shown in Fig.5, in which the electrons have discrete energy states. E0 is called the "ground state" and E1, E2, etc., are called "excited states".
In order for an electron to switch from E0 to E1, light with an energy of (E1 - E0) must strike the electron. This is the "absorption" of light. Electrons have particular energy levels,and rays of ultraviolet and visible light have the energy to change the energy states of the electrons.

Because the higher energy state, E1, is unstable, the electron soon returns to the ground state, E0. The energy discharged when the electron returns from E0 to E1 (E1 - E0) is converted to heat. If, for some reason, it is not converted to heat, the energy is discharged as light. The phenomenon of light emission is well known as fluorescence or phosphorescence.

In relation to quantitative measurement performed with spectroscopy, the consequence of this phenomenon is that there is a large amount of absorption if a large number of target molecules exist in a solution, and only a small amount of absorption if there is only a small number of target molecules.
Obtaining the quantity, and thereby the concentration, of a substance from the degree of absorption is the fundamental principle behind quantitative measurement.

The graph obtained by using the horizontal axis to represent the wavelength and the vertical axis to represent the degree of absorption is called an "absorption spectrum". The degree of absorption is expressed in terms of the unit "absorbance"(Abs). Fig.6 shows an absorption spectrum of β-carotene solution obtained with the Shimadzu UV-2550 UV-VIS spectrophotometer (Fig.7). β-carotene is the principle substance in carrots that gives them their color. As shown in Fig. 6, mainly blue and purple light of wavelengths in the range of 400 to 500 nm is absorbed. Because the visible light that reaches the eye of the observer consists of a mixture of the green and red components that are left over, carrots, which contain a large amount of β-carotene, appear to have a yellowred
Fig.6 Absorption Spectrum of β-carotene/Fig.7 Shimadzu UV-2550 UV-VIS Spectrophotometer

3. Summary

Here, we have looked at the properties of light and the way that it is absorbed, two fundamental aspects of the operation of a UV-VIS spectrophotometer. In the field of spectroscopy, in addition to UV-VIS spectrophotometers, there are various other types of spectroscopic measurement devices, such as infrared spectrophotometers, atomic absorption photometers, Raman spectrophotometers, and fluorescence spectrophotometers, all of which perform distinctive types of analysis. Using these devices selectively makes it possible to obtain various types of information about samples from different perspectives. In the next volume, we will describe the structure of a UV-VIS spectrophotometer.
1) Kanji Kihone: Measuring Light, Chapter 10
(Edited by the Illuminating Engineering Institute of Japan,Published by Nippon Riko Shuppankai, 1993), p. 172
2) Akira Harajima: Elementary Quantum Mechanics
(Shokabo, 1987), p. 3
3) Ryuzo Abe: Introduction to Quantum Mechanics
(Iwanami Shoten, Publishers, 1987), p. 31

Rabu, 20 November 2013

Uji Senyawa Antioksidan dengan Metode Difenilpikril hidrazil

Antioksidan merupakan zat yang mampu memperlambat atau mencegah terjadinya proses oksidasi1. Dalam konsentrasi yang rendah, zat ini dapat memperlambat dan mencegah terjadinya proses oksidasi yang menyebabkan terbentuknya radikal bebas dan berakibat terjadinya kerusakan protein dan asam nukleat dan memicu berbagai jenis penyakit seperti kanker dan penuaan dini.
Yang termasuk senyawa antioksidan adalah alkaloid, fenolik, polifenol, vitamin E, vitamin C, β-Karoten, EGCG, dan sebagainya. Selain itu, juga terdapat enzim yang memiliki kemampuan sebagai antioksidan seperti Superoksida Dimustase (SOD), katalase, ataupun glutation dismustase. Senyawa-senyawa ini terkandung dalam makanan dan minuman, suplemen makanan atau farmasi, ataupun kosmetik.
Dalam laboratorium uji, untuk melakukan assay atau uji aktivitas antioksidan, dikenal metode DPPH (Difenilpikril hidrazil). Zat ini berperan sebagai electron scavenger (penangkap elektron) atau hydrogen radical scavenger (penangkap radikal hidrogen bebas). Hasilnya adalah molekul yang bersifat dimagnetik dan stabil2. Jika suatu senyawa antioksidan direaksikan dengan zat ini maka senyawa antioksidan tersebut akan menetralkan radikal bebas dari DPPH. Pengukuran aktivitas antioksidan dilakukan dengan inkubasi DPPH dengan ekstrak antioksidan selama 30 menit sehingga menghasilkan larutan yang berwarna kuning kemudian dilakukan pengukuran panjang gelombang pada 517 nm. Reaksi yang terjadi adalah sebagai berikut3:
(DPPH) + (H—A) → DPPH—H + (A)
Ungu                         Kuning

H-A adalah senyawa antioksidan yang akan diuji.

Aktivitas antioksidan diperoleh dari nilai absorbansi yang selanjutnya akan digunakan untuk menghitung persentase inhibisi 50% (IC50) yang menyatakan konsentrasi senyawa antioksidan yang menyebabkan 50% dari DPPH kehilangan karakter radikal bebasnya. Semakin tinggi kadar senyawa antioksidan dalam sampel maka akan semakin rendah nilai IC50.
Merck Biosciences memproduksi DPPH yang digunakan dalam uji aktivitas senyawa antioksidan dengan bentuk padatan berwarna hitam (pelarutan dengan DMF atau etanol akan menghasilkan warna ungu kehitaman), memiliki kemurnian ≥ 90% dan larut dalam DMF atau etanol. Tersedia dengan kemasan 50 mg (300267-50MG) dengan brand Calbiochem®.

Spesifikasi Spektrofotometer UV-1800 Shimadzu di Laboratorium Kimia Farmasi UII

UV-VIS Spectrophotometer

Wavelength range 190 to 1100nm
Spectral bandwidth 1nm (190 to 1100nm)
Wavelength display 0.1-nm increments
Wavelength setting 0.1-nm increments (1-nm increments when setting scanning range )
Wavelength accuracy ±0.1nm at 656.1nm D2
±0.3nm (190 to 1100nm)
Wavelength repeatability ±0.1nm
Stray light less than 0.02% NaI at 220nm, NaNO2 at 340nm
less than 1.0% KCl at 198nm
Photometric system Double Beam
Photometric range Absorbance: -4 to 4 Abs
Transmittance: 0% to 400%
Photometric accuarcy ±0.002 Abs (0.5Abs)
±0.004 Abs (1.0Abs)
±0.006 Abs (2.0Abs)
Photometric repeatability less than ±0.001 Abs (0.5Abs)
less than ±0.001 Abs (1Abs)
less than ±0.003 Abs (2.0Abs)
Baseline stability less than 0.0003 Abs/H at 700nm
(one hour after light source turned ON)
Baseline flatness within ±0.0006 Abs
(190 to 1100nm,one hour after light source turned ON)
Noise level Within 0.00005 Abs RMS value (at 700nm)
Dimensions (W×D×H) 450(W) x 490(D) x 270(H)
Weight 15kg
Printers DPU, ESC/P, PCL printers, USB I/F Windows-compliant printers are available with USB memory and PC software
Memory USB memory (option) Saved as text and UVPC file
Performance for PC USB memory+UVProbe (standard) Win XP

Identifikasi Obat Palsu

Menurut Peraturan Menteri Kesehatan Nomor 10101 tahun 2008 tentang Registrasi Obat, yang dikategorikan sebagai Obat Palsu adalah obat yang diproduksi oleh yang tidak berhak berdasarkan peraturan perundang-undangan yang berlaku atau produksi obat dengan penandaan yang meniru identitas obat lain yang telah memiliki izin edar. 

Ada lima macam obat dikatagorikan palsu, yaitu :

  1. Produk mengandung bahan berkhasiat dengan kadar yang memenuhi syarat, diproduksi, dikemas dan diberi label seperti produk aslinya, tetapi bukan dibuat oleh pabrik aslinya tanpa adanya ijin/ lisensi dari pabrik aslinya/ pemegang ijin merk.
  2. Produk obat yang mengandung bahan berkhasiat dengan kadar yang tidak memenuhi persyaratan yang telah ditetapkan.
  3. Produk dibuat dengan bentuk dan kemasan seperti produk asli, tetapi tidak mengandung bahan berkhasiat.
  4. Produk yang menyerupai produk asli, tapi mengandung bahan berkhasiat yang berbeda.
  5. Produk yang diproduksi tidak berijin.
Produk impor tidak resmi yang tak memiliki izin edar dari Kemenkes cq Badan POM RI sesuai dengan Peraturan Menkes No 949/Menkes/SK/VI/2000.

Untuk mengetahui apakah obat yang beredar di warung warung termasuk obat palsu atau tidak,  dapat dilakukan di Laboratorium Kimia Farmasi UII.

Cara uji sederhana adalah dengan pemeriksaan sesuai Farmakope Indonesia tentang jenis obat yang beredar.

Untuk identifikasi dan penentuaan kadar zat aktif dapat di lakukan dengan metode spektrofotometri maupun KCKT.

ada hal menarik tentang obat kadaluwarsa yang di edarkan di warung warung kecil setelah di uji lab, ya namanya obat yang telah kadaluwarsa tentu mengalami pengurangan kadar zat aktif dan perubahan komposisi.

Minggu, 23 Desember 2012


Suggestions for Cleaning

Good laboratory technique demands clean glassware, because the most carefully executed piece of work may give an erroneous result if dirty glassware is used. In all instances, glassware must be physically clean; it must be chemically clean; and in many cases, it must be bacteriologically clean or sterile. All glassware must be absolutely grease-free. The safest criteria of cleanliness is uniform wetting of the surface by distilled water. This is especially important in glassware used for measuring the volume of liquids. Grease and other contaminating materials will prevent the glass from becoming uniformly wetted. This in turn will alter the volume of residue adhering to the walls of the glass container and thus affect the volume of liquid delivered. Furthermore, in pipets and burets, the meniscus will be distorted and the correct adjustments cannot be made. The presence of small amounts of impurities may also alter the meniscus.


Wash labware as quickly as possible after use. If a thorough cleaning is not possible immediately, put glassware to soak in water. If labware is not cleaned immediately, it may become impossible to remove the residue.
Most new glassware is slightly alkaline in reaction. For precision chemical tests, new glassware should be soaked several hours in acid water (a 1% solution of hydrochloric or nitric acid) before washing.
Brushes with wooden or plastic handles are recommended as they will not scratch or abrade the glass surface.
Glassware Cleaners
When washing, soap, detergent, or cleaning powder (with or without an abrasive) may be used. Cleaners for glassware include Alconox®, Dural®,M&H®, Lux®, Tide® and Fab®. The water should be hot. For glassware that is exceptionally dirty, a cleaning powder with a mild abrasive action will give more satisfactory results. The abrasive should not scratch the glass. During the washing, all parts of the glassware should be thoroughly scrubbed with a brush. This means that a full set of brushes must be at hand-brushes to fit large and small test tubes, burets, funnels, graduates and various sizes of flasks and bottles. Motor driven revolving brushes are valuable when a large number of tubes or bottles are processed. Do not use cleaning brushes that are so worn that the spine hits the glass. Serious scratches may result. Scratched glass is more prone to break during experiments. Any mark in the uniform surface of glassware is a potential breaking point, especially when the piece is heated. Do not allow acid to come into contact with a piece of glassware before the detergent (or soap) is thoroughly removed. If this happens, a film of grease may be formed.

Safe Use of Chromic Acid

If glassware becomes unduly clouded or dirty or contains coagulated organic matter, it must be cleansed with chromic acid cleaning solution1. The dichromate should be handled with extreme care because it is a powerful corrosive and carcinogen.
When chromic acid solution is used the item may be rinsed with the cleaning solution or it may be filled and allowed to stand. The length of time it is allowed to stand depends on the amount of contamination on the glassware. Relatively clean glassware may require only a few minutes of exposure; if debris is present, such as blood clots, it may be necessary to let the glassware stand all night. Due to the intense corrosive action of the chromic acid solution, it is good practice to place the stock bottle, as well as the glassware being treated, in flat glass pans or pans made from lead or coated with lead, or plastic polymer pans determined compatible with the concentration of chromic acid you are using. Extra care must be taken to be sure chromic acid solution is disposed of properly.
Special types of precipitates may require removal with nitric acid, aqua regia or fuming sulfuric acid. These are very corrosive substances and should be used only when required.

Removing Grease

Grease is best removed by boiling in a weak solution of sodium carbonate. Acetone or any other fat solvent may be used. Strong alkalis should not be used. Silicone grease is most easily removed by soaking the stopcock plug or barrel for 2 hours in warm decahydronaphthalene.
Drain and rinse with acetone or use fuming sulfuric acid for 30 minutes. Be sure to rinse off all of the cleaning agents.


It is imperative that all soap, detergents and other cleaning fluids be removed from glassware before use. This is especially important with the detergents, slight traces of which will interfere with serologic and cultural reactions.
After cleaning, rinse the glassware with running tap water. When test tubes, graduates, flasks and similar containers are rinsed with tap water, allow the water to run into and over them for a short time, then partly fill each piece with water, thoroughly shake and empty at least six times. Pipets and burets are best rinsed by attaching a piece of rubber tubing to the faucet and then attaching the delivery end of the pipets or burets to a hose, allowing the water to run through them. If the tap water is very hard, it is best to run it through a deionizer before using.
Rinse the glassware in a large bath of distilled water. Rinse with distilled water. To conserve distilled water, use a five gallon bottle as a reservoir. Store it on a shelf near your clean-up area. Attach a siphon to it and use it for replenishing the reservoir with used distilled water.
For sensitive microbiologic assays, meticulous cleaning must be followed by rinsing 12 times in distilled water.

Sterilizing Contaminated Glassware

Glassware which is contaminated with blood clots, such as serology tubes, culture media, petri dishes, etc., must be sterilized before cleaning. It can best be processed in the laboratory by placing it in a large bucket or boiler filled with water, to which 1-2% soft soap or detergent has been added, and boiled for 30 minutes. The glassware can then be rinsed in tap water, scrubbed with detergent, rinsed again.
You may autoclave glassware or sterilize it in large steam ovens or similar apparatus. If viruses or spore-bearing bacteria are present, autoclaving is absolutely necessary.

Handling and Storing

To prevent breakage when rinsing or washing pipets, cylinders or burets, be careful not to let tips hit the sink or the water tap.
Dry test tubes, culture tubes, flasks and other labware by hanging them on wooden pegs or placing them in baskets with their mouths downward and allowing them to dry in the air; or place them in baskets to dry in an oven2. Drying temperatures should not exceed 140°C. Line the drying basket with a clean cloth to keep the vessel mouths clean.
Dry burets, pipets and cylinders by standing them on a folded towel. Protect clean glassware from dust. This is done best by plugging with cotton, corking, taping a heavy piece of paper over the mouth or placing the glassware in a dust-free cabinet.
Store glassware in specially designed racks. Avoid breakage by keeping pieces separated.
Do not store alkaline liquids in volumetric flasks or burets. Stoppers or stopcocks may stick.

Proper care and handling of Pyrex® and PyrexPlus® labware will greatly increase its life and increase the safety of your work place.

PyrexPlus® labware can be successfully sterilized using liquids or dry cycle sterilization which involves no vacuum or low vacuum (<5 inches Hg).
Recommended cycles for automated autoclaves are:
Autoclave Type
Autoclave Cycle
CAUTION: Always autoclave vessels with loose caps or closures.
Steam sterilization time should not exceed 15 minutes at 121°C (250°F). Drying time should not exceed 15 minutes at 110°C (230°F). The actual cavity temperature of the autoclave should be checked to be sure the autoclave temperature does not exceed the recommended sterilization and drying temperature.
Should the coating appear clouded due to dissolved moisture, simply let dry overnight at room temperature or briefly heat to 110°C (230°F).
As is common practice, clean all glassware before use. Any non-abrasive glassware detergent may be used for hand or automatic dishwasher cleaning. If using a dishwasher or glassware dryer, care should be taken to be sure the drying temperature does not exceed 110°C (230°F). Exposure to dry heat should be minimized.
Avoid brushes and cleaning pads which could abrade the glass or damage the coating. If using a chromic acid cleaning solution minimize contact of the solution with the coating.
Labeling and Marking:
Use water-based markers for temporary marking or labeling of the PyrexPlus® labware coating. Solvent-based markers, dyes and stains cannot be removed from the coating.
NOTE: A slight “plastic” odor may be detected when handling PyrexPlus® labware. This is due to additives in the plastic coating which are responsible for its superior performance. The odor is normal and will not affect the inertness of the inside borosilicate glass surface.

Remove the stopcock or rubber tip and wash the buret with detergent and water. Rinse with tap water until all the dirt is removed. Then rinse with distilled water and dry. Wash the stopcock or rubber tip separately. Before a glass stopcock is placed in the buret, lubricate the joint with stopcock lubricant. Use only a small amount of lubricant. Burets should always be covered when not in use.

Culture Tubes:
Culture tubes which have been used previously must be sterilized before cleaning. The best method for sterilizing culture tubes is by autoclaving for 30 minutes at 121°C (15 p.s.i. pressure). Media which solidifies on cooling should be poured out while the tubes are hot. After the tubes are emptied, brush with detergent and water, rinse thoroughly with tap water, rinse with distilled water, place in a basket and dry.
If tubes are to be filled with a media which is sterilized by autoclaving, do not plug until the media is added. Both media and tubes are thus sterilized with one autoclaving.
If the tubes are to be filled with sterile media, plug and sterilize the tubes in the autoclave or dry air sterilizer before adding the media.

Dishes and Culture Bottles:
Sterilize and clean as detailed under Culture Tubes. Wrap in heavy paper or place in a petri dish can. Sterilize in the autoclave or dry air sterilizer.

Place pipets, tips down, in a cylinder or tall jar of water immediately after use. Do not drop them into the jar. This may break or chip the tips and render the pipets useless for accurate measurements. A pad of cotton or glass wool at the bottom of the jar will help to prevent breaking of the tips. Be certain that the water level is high enough to immerse the greater portion or all of each pipet. The pipets may then be drained and placed in a cylinder or jar of dissolved detergent or, if exceptionally dirty, in a jar of chromic acid cleaning solution. After soaking for several hours, or overnight, drain the pipets and run tap water over and through them until all traces of dirt are removed. Soak the pipets in distilled water for at least one hour. Remove from the distilled water, rinse, dry the outside with a cloth, shake the water out and dry.
Blood Cell Count Diluting Pipets:
After use, rinse thoroughly with cool tap water, distilled water, alcohol, or acetone, and then ether. Dry by suction. Do not blow into the pipets as this will cause moisture to condense on the inside of the pipet.
To remove particles of coagulated blood or dirt, a cleaning solution should be used. One type of solution will suffice in one case, whereas a stronger solution may be required in another. It is best to fill the pipet with the cleaning solution and allow to stand overnight. Sodium hypo chlorite (laundry bleach) or a detergent may be used. Hydrogen peroxide is also useful. In difficult cases, use concentrated nitric acid. Some particles may require loosening with a horse hair or piece of fine wire. Take care not to scratch the inside of the pipet.

Automatic Pipet Washers:
Where a large number of pipets are used daily, it is convenient to use an automatic pipet washer. Some of these, made of metal, can be connected directly by permanent fixtures to the hot and cold water supplies. Others, such as those made with polyethylene, can be attached to the water supplies by rubber hose. Polyethylene baskets and jars may be used for soaking and rinsing pipets in chromic acid cleaning solution. Electrically heated metallic pipet dryers are also available.
After drying, place pipets in a dust-free drawer. Wrap serologic and bacteriologic pipets in paper or place in pipet cans and sterilize in the dry air sterilizer. Pipets used for transferring infectious material should have a cotton plug placed in the top end of the pipet before sterilizing. The plug will prevent the material being measured from being drawn accidentally into the pipetting device.

Serological Tubes:
Serological tubes should be chemically clean, but need not be sterile. However, specimens of blood which are to be kept for some time at room temperature should be collected in a sterile container. It may be expedient to sterilize all tubes.
To clean and sterilize tubes containing blood, discard the clots in a waste container and place the tubes in a large basket. Put the basket, with others, in a large bucket or boiler. Cover with water, add a fair quantity of soft soap or detergent and boil for 30 minutes. Rinse the tubes, clean with a brush, rinse and dry with the usual precautions.
It is imperative when washing serological glassware that all acids, alkali and detergents be completely removed. Acids, alkalis and detergents in small amounts interfere with serologic reactions. Serologic tubes and glassware should be kept separate from all other glassware and used only for serologic procedures.

Slides and Cover Glass:
It is especially important that microscope slides and cover glass used for the preparation of blood films or bacteriologic smears be perfectly clean and free from scratches. Slides should be washed, placed in glacial acetic acid for 10 minutes, rinsed with distilled water and wiped dry with clean paper towels or cloth. Once the slides have been washed, place them in a wide jar of alcohol. As needed, remove from the jar and wipe dry. If the slides are dry stored, wash them with alcohol before use.

1.  Chromic acid cleaning solution — Use powdered commercial or technical grade sodium dichromate or potassium dichromate. If the compound is in the form of crystals, grind to a fine powder in a mortar. To 20 grams of the powder in a liter beaker, add a little water, sufficient to make a thin paste. Slowly add approximately 300mL of commercial concentrated sulfuric acid, stirring well. Transfer to a glass-stoppered bottle.
Larger amounts can be made in the same proportions. Use the clear supernatant solution. Chromic acid solution can be used repeatedly until it begins to turn a greenish color. Dispose of in accordance with appropriate regulations. Dilute with large volumes of water before discarding, or carefully neutralize the diluted solution with sodium hydroxide. Chromic acid solution is strongly acidic and will burn the skin severely. Use care in handling it.


Chemical Grade Definitions

Chemical grade definitions from highest to lowest purity...

1. A.C.S.

A chemical grade of highest purity and meets or exceeds purity standards set by American Chemical Society (ACS).

2. Reagent

High purity generally equal to A.C.S. grade and suitable for use in many laboratory and analytical applications.

3. U.S.P.

A chemical grade of sufficient purity to meet or exceed requirements of the U.S. Pharmacopeia (USP); acceptable for food, drug, or medicinal use; may be used for most laboratory purposes.

4. N.F.

A grade of sufficient purity to meet or exceed requirements of the National Formulary (NF).

5. Lab

A chemical grade of relatively high quality with exact levels of impurities unknown; usually pure enough for educational applications. Not pure enough to be offered for food, drug, or medicinal use of any kind.

6. Purified

Also called pure or practical grade, and indicates good quality chemicals meeting no official standard; can be used in most cases for educational applications. Not pure enough to be offered for food, drug, or medicinal use of any kind.

7. Technical

Good quality chemical grade used for commercial and industrial purposes. Not pure enough to be offered for food, drug, or medicinal use of any kind