CN105738522A - Method for detecting squalene contained in leaf tobacco based on high performance liquid chromatography - Google Patents
Method for detecting squalene contained in leaf tobacco based on high performance liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for detecting squalene contained in leaf tobacco based on high performance liquid chromatography. As for a leaf tobacco sample, trichloromethane serves as an extraction agent, Soxhlet extraction with scientific and reasonable process conditions is combined, high extraction efficiency is guaranteed, and high performance liquid chromatography detection is performed after silica gel purification treatment. According to the method disclosed by the invention, high performance liquid chromatography detection conditions are further optimized, chromatographic peak separation is excellent, the method has the advantages of accuracy, rapidness, high sensitivity, high reproducibility, high recovery rate and the like, and qualitative and quantitative requirements are met.
Description
Technical field
The present invention relates to the detection technique field of composition in Nicotiana tabacum L., detect the method for contained Squalene in Nicotiana tabacum L. more particularly, to a kind of based on high performance liquid chromatography.
Background technology
Squalene is find from the liver oil of shark at first, and within 1914, being named as Squalene, its chemical name is 2,6,10,15,19,23--hexamethyl--2,6,10,14,18,22--tetracosa carbon six alkene, belong to open chain triterpenoid compound.Squalene has superoxide dismutase (SOD) activity in raising human body, enhancing body non-oxidizability and immunocompetence, improves the different physiological roles such as sexual function, defying age, resisting fatigue, antitumor, is a kind of avirulent bioactive substance with the effect of preventing and curing diseases.Research recently finds that human body is also had Detoxication, the fat-soluble toxins in removable tissue by Squalene.In April, 2014, State Tobacco Monopoly Bureau issue " about " accelerate Tar technological achievement popularization and application notice (domestic cigarettes do comprehensive [2014] No. 218), proposition to strengthen the application dynamics of Harm reduction techniques technology key special subjects achievement in research, and Counter-techniques measure will be taked to accelerate Tar paces.
The applicant is at the ecological research work accumulated with harm reduction of Nicotiana tabacum L. Natural antioxidant Squalene, find that Squalene can optionally reduce the harmful components such as benzopyrene in flue gas, crotonic aldehyde, NH3, reduce Medicated cigarette hazard index, simultaneously Squalene has the gas phase removed in flue gas and phase Free Radicals function and has unique physiological function due to Squalene, and developing Squalene at present has become the focus that scientific and technological circle are studied.
But, at present both at home and abroad but without the detection method that Squalene in the Nicotiana tabacum L. being referred to is qualitative and quantitative, therefore, the detection method setting up Squalene in flue-cured tobacco qualitative and quantitative at tobacco business is forward-looking, is also very necessary.
Summary of the invention
The technical problem to be solved in the present invention is to fill up the detection technique deficiency of Squalene in existing Nicotiana tabacum L., it is provided that detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography.
Technical scheme is achieved by the following technical programs:
The present invention is with methanol and acetonitrile for mobile phase, adopt the column chromatography separation technology of the high-effective fixed phase being filled with particle diameter 5 μm, optimize high performance liquid chromatography testing conditions, sample after the inventive method pre-treatment is injected high performance liquid chromatograph and carries out chromatographic isolation, with ultraviolet absorption detector, the absorption value of detection Squalene, adopts external standard method, according to its content of absworption peak areal calculation；
Specifically, it is provided that a kind of based on the method for contained Squalene in high performance liquid chromatography detection Nicotiana tabacum L., comprise the following steps:
S1. sample pre-treatments obtains sample liquid, obtains sample liquid to be measured after processing through silica gel purification；
S2. preparing standard solution, Criterion curve；
S3. detection: pipette samples liquid to be measured injects high performance liquid chromatograph and carries out chromatographic isolation, detects Squalene with ultraviolet absorption detector；
S4. external standard method is adopted, according to its content of absworption peak areal calculation；
Wherein, sample pre-treatments described in step S1 comprises the following steps:
S11. tobacco leaf chopping, using chloroform as Extraction solvent, obtains extracting solution through surname extraction；
S12. by extracting solution through methanol: the extractant that the volume ratio of water is 70:30 extracts, collect extract, rotate and be evaporated near doing, then dissolve with acetonitrile, cross 0.22 μm of organic mesentery, collect filtrate and obtain sample liquid；
S13. being processed by too small for step S12 gained sample liquid column purification, adopt wet method dress post, little column material is 0.06g silica gel and appropriate anhydrous sodium sulfate；
Described in step S3, the condition of detection is:
Agilent1100 high performance liquid chromatograph HPLC joins UV-detector；
Chromatographic column: C18,5 μm, 4.6 × 250mm；
Column temperature: 30 DEG C；
Mobile phase type: methanol: acetonitrile, volume ratio is 75/25；
Flow rate of mobile phase: 1mL/min；
Detection wavelength is: 210nm；
Sample size: 10 μ L.
Preferably, the method for preparing standard solution described in step S2 is accurately to weigh 0.1g Squalene standard substance, is accurate to 0.0001g, is settled to 100mL and is configured to the Squalene standard solution of 1000mg/L after dissolving with acetonitrile, and fully vibration shakes up.
Preferably, described in step S2, the method for the foundation of Squalene standard curve is: adopt serial dilutions, be configured to respectively concentration be 0.002,0.01,0.05,0.2, the Squalene standard solution of 0.5mg/mL is in high performance liquid chromatography sample detection；With sample introduction concentration for abscissa, peak area is vertical coordinate Criterion curve, obtains the equation of linear regression of Squalene concentration and respective peaks area.
Preferably, tobacco shred, before adding chloroform, is first carried out vortex process by step S11, and it is vortex 1min that described vortex processes, static 10min.
Preferably, the condition that described vortex processes is 2000r/min.
Preferably, the consumption of chloroform described in step S11 is 5.0g tobacco shred according to the ratio with tobacco shred: 200mL chloroform is determined.
Preferably, the condition of described surname extraction described in step S11 is 40 DEG C of water-baths, refluxes 9 hours.
Preferably, the consumption of extractant described in step S12 is determined than for 1:1 with extracting liquid volume according to it.
Preferably, the number of times extracted described in step S12 is 3 times.
The present invention adds appropriate anhydrous sodium sulfate in cleanser and can prevent solution from rinsing silica gel well, causes uneven.Preferably, anhydrous sodium sulfate described in step S13 is 0.5g.
The method have the advantages that
Now there are some researches show, containing nearly 5000 kinds of compounds in the flue gas after cigarette burning, carry out chemical analysis according to the chloroform extract of Nicotiana tabacum L., record and wherein have 300 Multiple components, and the content of Squalene is very low, it be detected fast and accurately, it is necessary to set up the detection method of scientific system.The present invention is directed to tobacco sample, establish a kind of Squalene detection high performance liquid chromatography suitable in Nicotiana tabacum L., adopt chloroform as Extraction solvent, in conjunction with adopting soxhlet extraction, ensure higher extraction efficiency, purified treatment then through silicagel column, when the science pretreatment technology of described sample liquid to be measured, optimize testing conditions further, specific aim solves impurity peaks in sample and processes problem, effectively play high performance liquid chromatography accurate, quickly, high-sensitive advantage, provide a kind of chromatographic peak for Squalene in detection Nicotiana tabacum L. and separate better, accurately, quickly, highly sensitive, favorable reproducibility, the detection method that the response rate is high, not only conform with qualitative requirement, and meet quantitative requirement.
Accompanying drawing explanation
Fig. 1 Squalene canonical plotting.
Fig. 2 Squalene standard sample chromatogram.
Fig. 3 chloroform extraction be not added with object sample chromatogram figure.
The interpolation object sample chromatogram figure of Fig. 4 chloroform extraction.
Fig. 5 Squalene (50mg/L) standard solution chromatogram.
Fig. 6 background sample chromatogram.
Fig. 7 background adds sample chromatogram figure.
Fig. 8 Squalene standard solution (5mg/L) chromatogram.
Fig. 9 purifies the chromatogram of sample without cleanser.
Figure 10 is through the chromatogram of silica gel purification sample.
Figure 11 purifies the chromatogram of sample through PSA (N-propyl group ethylenediamine solid-phase adsorbent).
Detailed description of the invention
The inventive method is further illustrated below in conjunction with the drawings and specific embodiments.Following embodiment and accompanying drawing being merely cited for property explanation, it is impossible to be interpreted as limitation of the present invention.Unless stated otherwise, the reagent raw material used in following embodiment is reagent raw material that is conventional commercial or that be either commercially available, and unless stated otherwise, the method and apparatus used in following embodiment is method and apparatus commonly used in the art.For convenience of description, the instrument used in the embodiment of the present invention and reagent illustrate as follows, but therefore do not limit the present invention.
Instrument:
(1) high performance liquid chromatograph HPLC (Agilent1100), is equipped with: degassed pump, quaternary pump, automatic sampler, column oven and UV-detector (Agilent company of the U.S.)
(2) shaking table agitator: SHZ-82AB type (high honour instrument manufacturing company limited of Jintan City of Jiangsu Province)
(3) platform balance: T-10000 type, d=0.1g (double; two outstanding brother (group) company limited of the U.S.)
(4) electronic balance: BSA224S type, d=0.1mg (Sai Duolisi scientific instrument (Beijing) company limited)
(5) rotary vacuum evaporator: RE52-99 type (Shanghai Yarong Biochemical Instrument Plant)
(6) vacuum filtration pump: SHZ-D III type (Yu Hua Instrument Ltd. of Gongyi City)
(7) ultrasonic cleaner: KQ-50 type (Kunshan Ultrasonic Instruments Co., Ltd.)
(8) vortex instrument: MS3BS25 (IKA company)
(9) thermostat water bath: 501 digital displays (Hong Hua instrument plant of Jintan City of Jiangsu Province)
(10) the general glass drying oven of various laboratorys
Reagent:
(1) 99.8% Squalene reference substance (medicine inspecting institute of China)
(2) methanol (chromatographically pure and analytical pure)
(3) acetonitrile (chromatographically pure)
(4) normal hexane (analytical pure)
(5) chloroform (chloroform, analytical pure)
(6) petroleum ether (analytical pure)
(7) 0.22 μm of organic microporous filter membrane (Britain originates in film)
(8) acetone (analytical pure)
(9) cleanser: PSA, C18, silica gel (welchrom company)
(10) tobacco shred (Guangxi China Tobacco Industrial LCC randomly draws)
Embodiment 1
The present embodiment provides the detection method of Squalene in a kind of Nicotiana tabacum L., comprises the following steps:
S1. sample pre-treatments obtains sample liquid to be measured；
S2. preparing standard solution, Criterion curve；
S3. detection: pipette samples liquid to be measured injects high performance liquid chromatograph and carries out chromatographic isolation, detects Squalene with ultraviolet absorption detector；
S4. external standard method is adopted, according to its content of absworption peak areal calculation.
Wherein, the method for sample pre-treatments described in step S1 is:
nullSample-pretreating method: take 0.05mL、10000mg/L Squalene standard specimen obtains external standard basal liquid in 10mL normal hexane，After tobacco leaf chopping，Take tobacco shred 5.0g and (take 0.05mL，10000mg/L Squalene standard specimen is in 10mL normal hexane，Then by 5.0g tobacco shred to wherein，Vortex instrument vortex 1min in 2000r/min，Static，It is naturally evaporated to dryness (about 24h)，Then wrap with the filter paper that diameter is 15cm) in apparatus,Soxhlet's，Use normal hexane，Chloroform，Acetone 200mL is respectively placed in round-bottomed flask，Apparatus,Soxhlet's is put in 40 DEG C of water-baths，Reflux 9 hours，Collect extracting solution，Collect extracting solution，Tobacco shred is rinsed with 10mL chloroform，United extraction liquid，After after taking merging, extracting solution crosses pillar，Rotary evaporation is near dry，Dissolve with 5mL chromatographic grade acetonitrile again，Cross 0.22 μm of organic mesentery，Obtain analyte sample fluid；Described pillar of crossing is to adopt wet method dress post, and little column material is 0.06g silica gel+appropriate anhydrous sodium sulfate；
The testing conditions of described detection is:
High performance liquid chromatograph HPLC (Agilent1100) joins UV-detector；
Chromatographic column: C18,5 μm, 4.6 × 250mm；
Column temperature: 30 DEG C；
Mobile phase type: methanol: acetonitrile (volume ratio is 75/25)；
Flow rate of mobile phase: 1mL/min；
Detection wavelength is: 210nm；
Sample size: 10 μ L.
Described in step S2, the method for the foundation of Squalene standard curve is: adopt serial dilutions, is configured to Squalene standard solution that concentration is 0.002,0.01,0.05,0.2,0.5mg/mL respectively in high performance liquid chromatography sample detection；Adopt quantified by external standard method, with sample introduction concentration (mg/L) for abscissa, peak area (UV S) is vertical coordinate (being shown in Table 1) Criterion curve, as shown in Figure 1, obtain the equation of linear regression of Squalene concentration and respective peaks area, described regression equation is y=40.092x+52.046, R2=0.9999.
The corresponding relation of table 1 Squalene concentration of standard solution and peak area
It is shown that the correlation coefficient of standard curve is 0.9999, method linear good, it is possible to for the calculating that the mensuration of detection limit and object are quantitative.
Squalene standard sample chromatogram, with chloroform extraction be not added with object sample chromatogram figure, with the interpolation object sample chromatogram figure of chloroform extraction, Squalene (50mg/L) standard solution chromatogram, background sample chromatogram, background add sample chromatogram figure see respectively shown in accompanying drawing 2～accompanying drawing 7.
Embodiment 2 detection method and property with favourable conditions checking test
For verifying the superiority of detection method and testing conditions further, carry out following experiment respectively:
The comparison (C18 post) of 2.1 different extractant extraction efficiencies
2.1.1 sample-pretreating method
Matched group (CK): take tobacco shred 5.0g (wrapping with the filter paper that diameter is 15cm) in apparatus,Soxhlet's, it is respectively placed in round-bottomed flask with each 200mL of Extraction solvent, apparatus,Soxhlet's is put in 40 DEG C of water-baths, reflux 9 hours, collect extracting solution, 30mL is taken from extracting solution, put in separatory funnel, add 30mL extract methanol-water (V:V=70:30) to extract, repeat 3 times, collect extract, rotate and be evaporated near doing, dissolve with 5mL chromatographic grade acetonitrile again, cross 0.22 μm of organic mesentery, to be measured.
nullAdd sample pre-treatments: take tobacco shred 5.0g and (take 0.05mL，10000mg/L Squalene standard specimen is in 10mL normal hexane，Then by 5.0g tobacco shred to wherein，Vortex instrument vortex 1min in 2000r/min，Static，It is naturally evaporated to dryness (about 24h)，Then wrap with the filter paper that diameter is 15cm) in apparatus,Soxhlet's，It is respectively placed in round-bottomed flask with each 200mL of Extraction solvent，Apparatus,Soxhlet's is put in 40 DEG C of water-baths，Reflux 9 hours，Collect extracting solution，30mL is taken from extracting solution，Put in separatory funnel，Add 30mL extract methanol-water (V:V=70:30) to extract，Repeat 3 times，Collect extract，Rotate and be evaporated near doing，Dissolve with 5mL chromatographic grade acetonitrile again，Cross 0.22 μm of organic mesentery，It is put under 4 DEG C of conditions and preserves，To be measured.
For exempting to repeat one by one, the present embodiment is using Extraction solvent normal hexane, chloroform or acetone as explanation.
2.1.2 different solvents extraction efficiency result of the comparison
Different solvents extraction efficiency result of the comparison is in Table 2.The extraction efficiency of chloroform is the highest as can be seen from Table 2.
The extraction efficiency of the different extractant of table 2 compares
In table, "-" represents " without adding or without the response rate "
2.1.3 the discussion of different solvents extraction efficiency comparative result
Utilize soxhlet extraction, different extractants are extracted the efficiency of Squalene in sample and compares, from the response rate of result it can be seen that the extraction efficiency of chloroform is the highest, reach 62.27%, taking second place of normal hexane, reach the worst of 36.36% acetone, reach 18.59%.
The comparison (C18 post) of 2.2 different mobile phase ratios
In order to improve analysis efficiency, the present embodiment experiment devises mobile phase methanol: the impact on appearance time of the acetonitrile different proportion, the design included but not limited to has: methanol: acetonitrile=50:50, methanol: acetonitrile=70:30, methanol: acetonitrile=90:10, methanol: acetonitrile=10:90, methanol: acetonitrile=85:15, methanol: acetonitrile=80:20, methanol: acetonitrile=30:70, methanol: acetonitrile=40:60, methanol: the different proportions such as acetonitrile=75:25.The separation case of different proportion appearance time and target peak and impurity peaks is shown in Table 3:
The impact that object is separated by the different mobile phase ratio of table 3 with impurity peaks
Optimal flow phase condition (C18 post) under optimal conditions: methanol: acetonitrile=75:25 (30min) → methanol 100% (19min).Flow velocity=1mL/min, detects wavelength=210nm, column temperature=30 DEG C.
Result of the test is shown in that the standard solution chromatogram of Squalene (50mg/L) shown in accompanying drawing 5 to accompanying drawing 7, background sample chromatogram, background add sample chromatogram figure.
When carrying out mobile phase ratio difference, the effect that in sample, object separates with impurity peaks is at methanol: during acetonitrile=75:25, effect is more satisfactory.
The comparison (C18 post) of 2.3 Different Extraction Method extraction efficiencies
Under above-mentioned different optimal conditions, chloroform, as extractant, carries out surname extraction, and when extracting with extractant methanol-water (V:V=70:30), the extraction efficiency of sample is greatly improved.Result is shown in Table 4:
The extraction efficiency of object is compared by table 4 Different Extraction Method
The comparison (C18 post) of 2.4 cleanser clean-up effects
2.4.1 sample-pretreating method
Background sample pre-treating method (CK silica gel): with reference to aforementioned processing methods, take 2mL extracting solution after united extraction liquid and rotate evaporation, dissolved with appropriate normal hexane, then after carrying out post (0.06g silica gel+0.5g anhydrous sodium sulfate----wet Xian fills post method), rotary evaporation is near dry, dissolve with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane, to be measured.The present invention adds appropriate anhydrous sodium sulfate in cleanser and can prevent solution from rinsing silica gel well, causes uneven (lower same).
Sample-pretreating method (silica gel): with reference to aforementioned processing methods, take after 2mL crosses pillar (0.06g silica gel+appropriate anhydrous sodium sulfate----wet Xian fills post method) after united extraction liquid, rotary evaporation is near dry, dissolve with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane, to be measured.
Background sample pre-treating method (CK is straight): with reference to aforementioned processing methods, takes 2mL extracting solution and rotates and be evaporated near dry, dissolve with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane after united extraction liquid, to be measured.
Sample-pretreating method (directly): with reference to aforementioned processing methods, takes 2mL rotary evaporation near dry, dissolves with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane after united extraction liquid, to be measured.
Background sample pre-treating method (CK PSA): with reference to aforementioned processing methods, take 2mL extracting solution after united extraction liquid and rotate evaporation, dissolved with appropriate normal hexane, then after carrying out post (0.06gPSA+0.5g anhydrous sodium sulfate----wet Xian fills post method), rotary evaporation is near dry, dissolve with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane, to be measured.
Sample-pretreating method (PSA): with reference to aforementioned processing methods, take after 2mL crosses pillar (the appropriate anhydrous sodium sulfate of 0.06gPSA+----wet Xian fills post method) after united extraction liquid, rotary evaporation is near dry, dissolve with 2mL chromatographic grade acetonitrile, cross 0.22 μm of organic system filter membrane, to be measured.
2.4.2 high-efficient liquid phase chromatogram condition (C18 post)
Methanol: acetonitrile=75:25 (30min) → methanol 100% (19min).Flow velocity=1mL/min, detects wavelength=210nm, column temperature=30 DEG C.
Result of the test is shown in shown in accompanying drawing 8 to accompanying drawing 11, wherein, accompanying drawing 8 is Squalene standard solution (5mg/L) chromatogram, and accompanying drawing 9 is the chromatogram purifying sample without cleanser, accompanying drawing 10 is the chromatogram through silica gel purification sample, and accompanying drawing 11 is the chromatogram purifying sample through PSA.By accompanying drawing 8 to accompanying drawing 11 it can be seen that in the sample that cleanser purifies impurity peaks more non-purified decrease many, the simultaneously clean-up effect of the silica gel good purification than PSA, determine with silica gel as cleanser it is optimal in the methods of the invention so demonstrating.
Claims (10)
1. one kind is detected the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography, it is characterised in that comprise the following steps:
S1. sample pre-treatments obtains sample liquid, obtains sample liquid to be measured after processing through silica gel purification；
S2. preparing standard solution, Criterion curve；
S3. detection: pipette samples liquid to be measured injects high performance liquid chromatograph and carries out chromatographic isolation, detects Squalene with ultraviolet absorption detector；
S4. external standard method is adopted, according to its content of absworption peak areal calculation；
Wherein, sample pre-treatments described in step S1 comprises the following steps:
S11. tobacco leaf chopping, using chloroform as Extraction solvent, obtains extracting solution through surname extraction；
S12. by extracting solution through methanol: the extractant that the volume ratio of water is 70:30 extracts, collect extract, rotate and be evaporated near doing, then dissolve with acetonitrile, cross 0.22 μm of organic mesentery, collect filtrate and obtain sample liquid；
S13. being processed by too small for step S12 gained sample liquid column purification, adopt wet method dress post, little column material is 0.06g silica gel and appropriate anhydrous sodium sulfate；
Described in step S3, the condition of detection is:
Agilent1100 high performance liquid chromatograph HPLC joins UV-detector；
Chromatographic column: C18,5 μm, 4.6 × 250mm；
Column temperature: 30 DEG C；
Mobile phase type: methanol: acetonitrile, volume ratio is 75/25；
Flow rate of mobile phase: 1mL/min；
Detection wavelength is: 210nm；
Sample size: 10 μ L.
2. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterized in that, the method of preparing standard solution described in step S2 is accurately to weigh 0.1g Squalene standard substance, it is accurate to 0.0001g, being settled to 100mL after dissolving with acetonitrile and be configured to the Squalene standard solution of 1000mg/L, fully vibration shakes up.
3. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterized in that, described in step S2, the method for the foundation of Squalene standard curve is: adopt serial dilutions, be configured to respectively concentration be 0.002,0.01,0.05,0.2, the Squalene standard solution of 0.5mg/mL is in high performance liquid chromatography sample detection；With sample introduction concentration for abscissa, peak area is vertical coordinate Criterion curve, obtains the equation of linear regression of Squalene concentration and respective peaks area.
4. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that tobacco shred, before adding chloroform, is first carried out vortex process by step S11, and it is vortex 1min that described vortex processes, static 10min.
5. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 4, it is characterised in that the condition that described vortex processes is 2000r/min.
6. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the consumption of chloroform described in step S11 is 5.0g tobacco shred according to the ratio with tobacco shred: 200mL chloroform is determined.
7. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the condition of described surname extraction described in step S11 is 40 DEG C of water-baths, refluxes 9 hours.
8. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the consumption of extractant described in step S12 is determined than for 1:1 with extracting liquid volume according to it.
9. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that the number of times extracted described in step S12 is 3 times.
10. detect the method for contained Squalene in Nicotiana tabacum L. based on high performance liquid chromatography according to claim 1, it is characterised in that anhydrous sodium sulfate described in step S13 is 0.5g.
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