<p class="jove_content"> 이 작품은 건강의 국립 연구소 부여 번호 NIH / NCRR P41 - RR 016,292 (ACERT에 대한)에 의해 지원되었다. </p>

<p class="jove_title"> 1. 자료 </p><p class="jove_content"> 이 작품에 사용된 모든 용매는 시약 등급 있었다. 스핀 N - tert - 부틸 - α - phenylnitrone (PBN)와 표준 스핀 라벨을 포함하여 트랩, 2,2,6,6 - tetramethyl - 1 - piperinyoxyl이 (템포) 시그마에서 얻은되었고 공급으로 사용되었습니다. 공장 산화 억제제는 Swanson, 주식 회사 미국에서 상업적으로 획득했다. </p><p class="jove_title"> 2. 자유 래디 칼의 담배 연기 및 분석의 준비 </p><ol><li> lycopene은 아세톤에 녹아있는 동안 필터에 산화 억제제를 소개하려면, 산화 억제제의 pycnogenol과 포도 종자 추출물 먼저, 95 % 에탄올에 용해되었다. 솔벤트 볼륨 항산화 용해도에 따라 달랐다. </li><li> 0.4의 산화 억제제의 양은 MG / 필터가 사용되었습니다. 그들은 다음 활성탄의 10 MG로 코팅했다. 이런 목적을 위해 활성 탄소 필터와 진공 하에서 건조 무산소 조건에서 항산화 솔루션 ~ 12 시간을 위해 흔들되었다. </lI&gt; <li> 산화 억제제는 다음 종래의 아세테이트 필터 (CA 필터)에 도입되었습니다. 이를 위해 필터를 두 조각으로 절단되었다. 항산화 필터 샌드위치 (그림 1A) - 코팅 공장 산화 억제제는 필터를 형성하기 위해 필터와 테이프의 조각으로 싼 두 조각 사이에 삽입되었습니다. 이 결합된 필터는 이후에 담배를 포함하는 담배 막대에 붙어 있 었지. 필터가없이 항산화 제외 항산화 필터와 같은 방식으로 만들어진 컨트롤이 추가되었습니다. </li><li> 전에 흡연 시뮬레이션하는 연구 담배는 압축을 푼 2 일 최소 포화 NaBr 솔루션을 사용하여 일정한 습도 환경 (20 ° C, 60 % 상대 습도)에 보관되었다. </li><li> 일상적인 분석을 위해 흡연 시뮬레이션이로 Fig.1과 같이 단일 포트 흡연 장치를 사용하여 주위 온도에서 공연되었다, 물 흡인기 또는 GAST DOA - P104 - BN의 진공 펌프 / 공기 압축기 (벤톤 하버, Mich) 연결로 구성되어 있습니다 스핀 - 트래핑하기오픈 한 쪽 끝에있는 T - 교차점을 통해 조립. 퍼프는 퍼프 사이에 열어 두었던 오픈 엔드를 연결해에 의해 수행되었다. 가스 흐름이 유량계에 의해 체크 ~ 2.2에 설정된 SCFH = 17.5 펌프와 통풍구 사이에 위치 밸브를 조정하여 ML / s의. </li><li> 연구 담배는 <sup>1과</sup> 비슷한 60 초마다 반복 이초의 기간 동안 35 ML의 퍼프 볼륨의 상태에 따라 훈제했다. 가스 상 자유 래디 칼의 양적 추정을위한 흡연 시뮬레이션으로 그림에 표시된 스핀 트래핑 시스템과 연계하여 수행되었다. 1. </li><li> 열 강한 퍼프 (35 ML / 퐁퐁)은 각 담배에 대한 촬영했다. 가스 상 자유 래디 칼은 캠브리지 필터 패드를 통해 MCS를 전달하여 수집된 후 스핀 트래핑 솔루션 (벤젠, 2.0 ML에서 0.05 M PBN)에 도입되었습니다. </li><li> 마지막 퍼프 후, 부풀어 오른 트래핑 솔루션은 동일한 벤젠과 초기 볼륨 (2.0 ML)로 재조정했다. 나누어지는는 ~ 25 안으로 옮겨졌 다가길이 0mm, 한쪽 끝에 봉인 3mm의 ID 유리관. </li><li> 트래핑 솔루션은 냉동 펌프 - 해동 절차를 사용 deoxygenated되었습니다. 그것은 액체 질소에 따라 냉동었고, 진공이 적용되었습니다. 그렇다면 그것은 함정에 가스 거품이 탈출 수 있도록, 아르곤 분위기 하에서 해동되었고, 다시 냉동. 이 사이클은 세 번 반복 후, 튜브했습니다 불꽃 - 밀봉 진공하에 추가적인 ESR 측정에 사용. 벤젠에 잘 용해됩니다 산소, 유기 래디 칼의 ESR 라인을 넓혀 때문에이 단계가 필요합니다. 탈산소 반응은 극적으로 신호 대 잡음 비율을 향상시킵니다. </li><li> X - 밴드 ESR 스펙트럼은 표준 조건에서 9.34 GHz의 주파수에서 Bruker EMX 분광계에 기록되었다. 대부분의 실험에 사용되는 분광계 설정되었습니다 : 센터 현장 3312.5G, 스캔 폭 80G, 변조 진폭 0.5G, 시간 상수 82 마이크로초, 검사 시간 40 초. </li><li> 스핀 트랩 부가물 어떤 경우에는 req 실험 조건에서 비교적 안정일반적으로 ~ 20 분 데려 25 쌓일, uired. 그러나, 12 시간 이후 벤젠 용액의 ESR 신호의 강도가 ~ 5 요소에 의해 감소​​. </li><li> 트래핑 adduct의 농도를 정량하기 위해 초기 처음 유도체 ESR 스펙트럼이 통합되었습니다. 그 결과 흡수 스펙트럼의 연기에 포함된 검댕 / 타르 제품에 따라 가능성이 가장 광범위한 일중 배경을 보여줍니다. <br /><br /> 뺄셈 후에이 배경의 트래핑 adduct의 분리 삼인승는 한 번 더 (그림 2) 통합되었습니다. </li></ol><p class="jove_title"> 3. 대표 결과 </p><p class="jove_content"> 담배 생산 연기 (기상)를 굽기에있는 자유 래디 칼의 대부분은 즉시하고 불안정하고 있습니다. 이러한 래디 칼을 관찰하기 위해 스핀 트랩 기술을 채용합니다. 그것은보다 안정되고 ESR (Fig.1)에 의해 감지 수있는 스핀 adduct로 그들을 변환하여 가스 상 자유 래디 칼을 캡처합니다. 0.05의 현재 연구에서 스핀 - 트랩 솔루션M PBN <sup>15을</sup> 구분하기가 어렵습니다 산소와 탄소 중심의 래디 칼의 혼합 아르 자유 래디 칼, 위상 연기 가스를 수집하기 위해 사용되었다. 우리의 경우에는, 그러나, 관찰 초미세 분리 상수 <sub>N</sub> = 13.7G와 <sub>H</sub> = 1.95G들이 주요 제품에 것을 제안, alkoxyl 자유 래디 칼 (RO.) <sup>2</sup> 부가물를 트래핑에 해당하는 값이 매우 유사합니다. 우리는 연기의 흐름에 습기로 인해 있다고 약한 ESR 신호 (그림 2) 초기 측정에서 관찰 낮은 재현성을 보여주었다. 이 문제를 해결하기 위해, 우리는 캠브리지 필터와 스핀 트랩 솔루션 사이에 액체 질소 (LN2) 트랩을 추가했습니다. <sub>에선이</sub> 함정 급속 냉동하여 MCS 흐름에서 물을 제거하고 유리관의 내벽에 캡처. 이것은 크게 ESR 신호를 개선하고 높은 재현성 결과를 (그림 3) 허용. </p><p class="jove_content"> 함정 자유 래디 칼의 금액을 사용하여 결정되었다참고 샘플. 컨트롤 샘플 산화 억제제없이, 템포 알려진 농도의 이중 적분 스펙트럼과의 이중 적분 스펙트럼의 비교에 의해 예상 벤젠의 전형적인 adduct 농도는 1.24 μm의 (Fig.2)되었습니다. 공기의 양이 흡연하는 동안 각각의 담배 통과 이후 ~ 350 ML, 이것은 ~ 7.1X10 <sup>-9</sup> M의 MCS의 가스 단계에서 자유 라디칼 농도에 대한 견적을 제공하고, 래디 칼의 총 숫자는 가스 단계에서 덫에 걸렸다 ~ 1.5X10 <sup>15</sup> 전체 담배. 가스 및 미립자 단계 모두 포함한 전체 담배의 연기에 자유 래디 칼의 총 금액에 대한 견적은 <sup>16</sup> ~ 10 자유 래디 칼 <sup>9했습니다.</sup> </p><p class="jove_content"> 메인 스트림 담배 연기의 가스 단계 자유 래디 칼에 식물 산화 억제제의 청소 효과의 다른 수준이 관찰되었다. 그들의 청소 요금은 그림에서 소개되었습니다. 4. Lycopene과 포도 씨앗 추출물은 가장 높은 속도를 보여주 낮은 반면어 속도는 pycnogenol (Fig.4)에 대한 관찰되었다. </p><p class="jove_content"><img alt="그림 1" src="/files/ftp_upload/3406/3406fig1.jpg" /><br /> <strong>그림 1.</strong> 스핀 트랩을 사용하여 주류 담배 연기 (MCS)에서 가스 위상 자유 래디 칼을 수집에 대한 개선 흡연 시뮬레이션 설계의 도표. MCS는 다음 CA 필터, 캠브리지 필터 (노란색 필터)를 통해 물 흡인기에 의해 그려진 및 H <sub>2</sub> O.을 제거하는 액체 질소 트랩 통과되었다 가스 상 제품은 마침내 함정을 스핀에 가서 스핀 트랩 솔루션을 통해 부풀어 오른 모양이었다. 식물 항산화는 MCS의 자유 래디 칼을 청소하기 위해 담배에 부착 (동그라미로 확대) 종래의 아세테이트 필터의 두 가지 사이에 위치했다. </p><p class="jove_content"><img alt="그림 2" src="/files/ftp_upload/3406/3406fig2.jpg" /><br /> <strong>그림 2.</strong> 벤젠의 스핀 - 트래핑의 adduct 농도에 대한 양적 평가는 첫 번째 integra에서 광범위한 배경 신호를 빼서 필요내 스펙트럼. 스핀 - 트래핑의 adduct에 대한 초미세 분리 매개 변수 <sub>H</sub> = 1.95 G, <sub>N</sub> = 13.7G입니다. </p><p class="jove_content"><img alt="그림 3" src="/files/ftp_upload/3406/3406fig3.jpg" /><br /> <strong>그림 3.</strong> LN2 트랩을 통해 전달이 상당히 MCS의 스핀 - 트래핑 얻은 ESR 신호의 품질을 향상시킵니다. </p><p class="jove_content"><img alt="그림 4" src="/files/ftp_upload/3406/3406fig4.jpg" /><br /> <strong>그림 4.</strong> MCS의 자유 래디 칼의 농도에 자연 산화 억제제의 효과. 상대 신호 농도는 다음과 같습니다 제어 - pycnogenol 100 % - 55 %, 포도 종자 추출물 - 12 %, lycopene - 10 %. </p>

<ol>
	<li>Baum, S. L., Anderson, I. G. M., Baker, R. R., Murphy, D. M., Rowlands, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+spin+resonance+and+spin+trap+investigation+of+free+radicals+in+cigarette+smoke:+development+of+a+quantification+procedure.">Electron spin resonance and spin trap investigation of free radicals in cigarette smoke: development of a quantification procedure.</a> <em style='font-style: italic'>Analytica Chimica. Acta</em>. <b>481</b>, 1-13 (2003).</li><li>Bluhm, A. L., Weinstein, J., Sousa, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+tobacco+smoke.">Free radicals in tobacco smoke.</a> <em style='font-style: italic'>Nature</em>. <b>229</b>, 500-500 (1971).</li><li>Church, D. F., Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-radical+chemistry+of+cigarette+smoke+and+its+toxicological+implications.">Free-radical chemistry of cigarette smoke and its toxicological implications.</a> <em style='font-style: italic'>Environ. Health. Perspect</em>. <b>64</b>, 111-126 (1985).</li><li>Deliconstantinos, G., Villiotou, V., Stavrides, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenging+effects+of+hemoglobin+and+related+heme+containing+compounds+on+nitric+oxide,+reactive+oxidants+and+carcinogenic+volatile+nitrosocompounds+of+cigarette+smoke.+a+new+method+for+protection+against+the+dangerous+cigarette+constituents.">Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. a new method for protection against the dangerous cigarette constituents.</a> <em style='font-style: italic'>Anticancer. Res</em>. <b>14</b>, 2717-2726 (1994).</li><li>Hecht, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke+carcinogens+and+lung+cancer.">Tobacco smoke carcinogens and lung cancer.</a> <em style='font-style: italic'>Journal of the National Cancer Institute</em>. <b>91</b>, 1194-1210 (1999).</li><li>Kodama, M., Kaneko, M., Aida, M., Inoue, F., Nakayama, T., Akimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+chemistry+of+cigarette+smoke+and+its+implication+in+human+cancer.">Free radical chemistry of cigarette smoke and its implication in human cancer.</a> <em style='font-style: italic'>Anticancer. Res</em>. <b>17</b>, 433-437 (1997).</li><li>Nair, A. K., Brandt, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+smoking+on+health+care+costs.">Effects of smoking on health care costs.</a> <em style='font-style: italic'>Journal of Oklahoma State Medical Association</em>. <b>93</b>, 245-250 (2000).</li><li>Nishizawa, M., Kohno, M., Nishimura, M., Kitagawa, A., Niwano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+Peroxyradicals+in+Cigarette+Smoke+and+the+Scavenging+Effect+of+Shikonin,+a+Naphthoquinone+Pigment.">Presence of Peroxyradicals in Cigarette Smoke and the Scavenging Effect of Shikonin, a Naphthoquinone Pigment.</a> <em style='font-style: italic'>Chem. Pharm. Bull</em>. <b>53</b>, 796-799 (2005).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+and+the+involvement+of+free+radical+reactions+in+chemical+carcinogenesis.">Cigarette smoke and the involvement of free radical reactions in chemical carcinogenesis.</a> <em style='font-style: italic'>Br. J. Cancer</em>. <b>8</b>, 19-23 (1987).</li><li>Pryor, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+radicals+and+the+role+of+free+radicals+in+chemical+carcinogenicity.">Cigarette smoke radicals and the role of free radicals in chemical carcinogenicity.</a> <em style='font-style: italic'>Environ. Health. Perspect</em>. <b>105</b>, 875-882 (1997).</li><li>Rozzi, N. I., Singh, R. K., Vierling, R. A., Watkins, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercritical+fluid+extraction+of+lycopene+from+tomato+processing+byproducts.">Supercritical fluid extraction of lycopene from tomato processing byproducts.</a> <em style='font-style: italic'>J. Agric. Food. Chem</em>. <b>50</b>, 2638-2643 (2002).</li><li>Shopland, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+use+and+its+contribution+to+early+cancer+mortality+with+a+special+emphesis+on+cigarette+smoking.">Tobacco use and its contribution to early cancer mortality with a special emphesis on cigarette smoking.</a> <em style='font-style: italic'>Environmental. Health. Perspectives</em>. <b>103</b>, 131-142 (1995).</li><li>Shrikhandle, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wine+products+with+health+benefits.">Wine products with health benefits.</a> <em style='font-style: italic'>Food Res. Int</em>. <b>33</b>, 469-474 (2000).</li><li>Valavanidis, A., Haralambous, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+by+electron+paramagnetic+resonance+of+free+radical+species+in+the+mainstream+and+sidestream+smoke+of+cigarettes+with+conventional+acetate+filters+and+'bio-filters'.">A comparative study by electron paramagnetic resonance of free radical species in the mainstream and sidestream smoke of cigarettes with conventional acetate filters and 'bio-filters'.</a> <em style='font-style: italic'>Redox. Rep</em>. <b>6</b>, 161-171 (2001).</li><li>Valavanidis, A., Vlachogianni, T., Fiotakis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+smoke:+involvement+of+reactive+oxygen+species+and+stable+free+radicals+in+mechanisms+of+oxidative+damage,+carcinogenesis+and+synergistic+effects+with+other+respirable+particles.">Tobacco smoke: involvement of reactive oxygen species and stable free radicals in mechanisms of oxidative damage, carcinogenesis and synergistic effects with other respirable particles.</a> <em style='font-style: italic'>International Journal of Environmental Research and Public Health</em>. <b>6</b>, 445-462 (2009).</li></ol>

<p class="jove_content"> 담배 연기에있는 다른 자유 라디칼 청소부의 효과의 신뢰성 추정은 자유 래디 칼의 양적 검출을위한 기술을 재현할 수 있어야합니다. 이전 <sup>1,</sup> 그것은 비 극성 용매에서 높은 농도 스핀 트랩 솔루션의 작은 볼륨이 담배 연기에서 자유 래디 칼을 트래핑에 가장 효과적인 것으로 나타났습니다. 담배 연기는 항상 유기 화합물의 연소 및 트래핑 용매에 끝날 수있는 담배의 잔류 수분에서 수증기가 포함되어 있습니다. PBN의 스핀 트랩 용액에 물이 혼합물은 크게 스핀 트래핑 부가물의 수명과 ESR 신호의 강도를 감소시킵니다. 가스 상 래디 칼의 일부 분율도 얼어붙은 표면에 갇혀있을 경우에도 액체 질소로 냉각 U 자형 튜브를 통하여 간단하게 통과 MCS하여 수분을 제거하면 크게, 우리의 실험에서 ESR 스펙트럼의 품질을 개선. </p><p class="jove_content"> t를 사용그의 기술은 우리가 담배 필터에 도입된 천연 화합물을 다른 급진 -​​ 소기의 상대적 효율성을 비교. 우리는 lycopene과 포도 씨앗 담배 필터로 통합 후 기상 MSC에서 자유 래디 칼의 90 %까지 청소하실 수 있습니다 즉시 추출 것으로 나타났습니다. 이러한 높은 청소 능력은 헤모글로빈과 shikonine <sup>8,14</sup> 같은 가장 효율적인보고 자유 라디칼 프로텍터 중에서이 저렴한 즉시 사용 가능한 천연 화합물을 차지한다. 그러나, 공부 천연 산화 억제제와 함께 로드된 실험의 담배 필터에 실온에서 스토리지 일주일 후 그들의 소기 용량의 눈에 띄는 부분을 잃었습니다. 이 문제를 해결하는 것은 lycopene과 상업 담배 필터의 포도 종자 추출물 미래의 애플 리케이션을 자극 수 있습니다. </p>

a protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

<p class="jove_content"> 흡연은 인간의 암와 관련된 것입니다. 이것은 폐암 사망자의 대부분은 흡연 <sup>5,6,7,12로</sup> 인해 발생하는 것이보고되었습니다. 담배 연기의 입자 단계에서 담배 tars 및 관련 제품이 발암성 및 mutagenic 관련 질병의 주요 원인이지만, 담배 연기도 <sup>9,10 물질들의</sup> 중요한 그룹으로 간주됩니다 자유 래디 칼의 상당 금액을 포함하고 있습니다. 자유 래디 칼은 손상 단백질 구조, lipids과 DNA 시퀀스에 의해 세포 성분을 공격하고 암 다양한 형태의 개발의 위험을 증가시킵니다. 흡입 급진파는 폐 <sup>3</sup> 담배 연기의 부정적인 건강 효과를 많은 기여 부가물을 생산합니다. 연구는 흡연 유발 손상의 위험을 감소하기 위해 담배 연기에 자유 래디 칼을 줄이기 위해 실시되었습니다. 이것은 헤모글로빈과 헴이 함유 화합물 것은 부분적으로 질산 산화, 반응을 청소 수있는 것으로보고되었습니다oxidants와 담배 연기의 <sup>4</sup> 발암성 휘발성 nitrosocompounds. 헤모글로빈과 활성탄은 자유 래디 칼을 청소하고 담배 연기 <sup>14에서</sup> 자유 래디 칼의 90 %까지 제거하는 데 사용된의 &#39;바이오 필터&#39;는 구성된. 그러나, 비용 비효율로 인해, 그것은 성공적으로 상용화되지 않았습니다. 또 다른 연구는 shikonin, 중국 한방 의학 <sup>8</sup> 구성 요소의 좋은 청소 효율을 보여주었다. 현재 연구에서는, 우리는 스핀 - 트래핑을 사용하여 주류 담배 연기에 가스상 자유 래디 칼의 청소 효과 (MCS)의 담배 연기 및 측정의 가스상 자유 래디 칼을 청소에 대한 담배 필터에 일반 자연 항산화 추출물을 도입을위한 프로토콜을보고 전자 스핀 공명 (ESR) 분광학 <sup>1,2,14.</sup> 우리는 lycopene과 담배 필터에 자신의 미래의 응용 프로그램을 가리킨 수있는 포도 종자 추출물의 높은 청소 능력을 보여주었다. 이 프로의 중요한 이점pective 청소부들은 각각 토마토 또는 와인 산업의 부산물 <sup>11,13에서</sup> 대량으로 얻을 수있다는 것입니다 </p>

Watch this Scientific Journal Video about A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke at JoVE.com

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

<p class="jove_content">스핀 - 트래핑 ESR 분광법은 공장 산화 억제제 lycopene, pycnogenol와 담배 연기의 가스 상에게 자유 래디 칼을 청소에 포도 종자 추출물의 효과를 연구하는 데 사용되었다.</p>

메인 스트림 담배 연기에 자유 래디 칼을 감지하고 가스 단계 청소를위한 프로토콜

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

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JoVE Journal

Bioengineering

19.3K 조회수.  CDCF-AOX Lab. 스핀 - 트래핑 ESR 분광법은 공장 산화 억제제 lycopene, pycnogenol와 담배 연기의 가스 상에게 자유 래디 칼을 청소에 포도 종자 추출물의 효과를 연구하는 데 사용되었다.

비디오: A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Identifying Alcohols

<h4><strong>Alcohols</strong></h4>
<p>Alcohols are organic compounds that are amongst the most recognizable and familiar, as they have wide-ranging applications and uses in everyday life. Alcohols are organic molecules containing a hydroxyl functional group connected to an alkyl or aryl group (ROH). If the hydroxyl carbon only has a single R group, it is known as primary alcohol. If it has two R groups, it is a secondary alcohol, and if it has three R groups, it is a tertiary alcohol. Like many other organic compounds, alcohols can also be aromatic by containing a benzene ring. The simplest aromatic alcohol is phenol.</p>
<p>Identifying an alcohol in organic chemistry can be achieved by exploiting the different properties of the types of alcohols. While various instrumentation analysis methods, such as nuclear magnetic resonance (NMR) can be used, other qualitative tests can also be utilized. By coupling these tests together, the identity of an alcohol can be determined in a similar fashion to the way aldehydes and ketones can be identified.</p>
<h4><strong>Ferric Chloride Test</strong></h4>
<p>One method to differentiate between an aliphatic alcohol and an aromatic alcohol is by using iron(III) chloride. The iron chloride compound gives the solution a red-orange appearance. In the presence of an aromatic alcohol, like phenol, the chloride atoms are replaced by the aromatic alcohol, changing the coordination property of the center iron atom. This changes the color of the solution to a purple color. Aliphatic alcohols will not react with iron(III) chloride and, thus, the solution remains red-orange.</p>
<h4><strong>Jones Test</strong></h4>
<p>The Jones test utilizes chromium trioxide in the presence of sulfuric acid to act as a powerful oxidizing agent. In the presence of the Jones&#39; reagent, a primary alcohol is first converted into an aldehyde and then into a carboxylic acid, while a secondary alcohol will be oxidized into a ketone. The oxidation state of chromium is the key to this test. Chromium is in the +6 oxidation state in the Jones&#39; reagent. The Cr(VI) complexes in the reagent give it its bright reddish, orange color.</p>
<p>In the process of the reaction, chromium is reduced from Cr(VI) to the +3 oxidation state &#8212; Cr(III). First, the alcohol and chromic acid form a chromate ester. Then, a base (H<sub>2</sub>O) cleaves the C-H bond of the alcohol, forming the carbonyl group while reducing Cr(VI) to Cr(IV). The carbon of the alcohol undergoes a 2-electron oxidation, and the Cr(IV) undergoes a 2-electron reduction, so this is a reduction-oxidation step.</p>
<p>Cr(IV) participates in further oxidation steps and is eventually reduced to Cr(III). Cr(III) is often present as hexaaquachromium(III) ions &#8212; [Cr(H<sub>2</sub>O)<sub>6</sub>]<sup>3+ </sup>&#8212; and Cr(III) complexes, where H<sub>2</sub>O molecules are replaced by one or more sulphate ions &#8212; [Cr(H<sub>2</sub>O)<sub>5</sub>(SO<sub>4</sub>)]<sup>+</sup>. These complexes give Cr(III) the characteristic green color.</p>
<p>Tertiary alcohols do not react with chromium, and thus no precipitate is made, keeping the solution orange. Therefore, the Jones test can help differentiate primary and secondary alcohols from tertiary alcohols.</p>
<h4><strong>Lucas Test</strong></h4>
<p>The Lucas test utilizes zinc(II) chloride in the presence of hydrochloric acid as a reagent. In the presence of an alcohol, the Lucas reagent will halogenate the alcohol, making an insoluble product in aqueous solutions. The reaction rate for this is highly dependent on the formation of a carbocation caused by the loss of the hydroxyl as water. A carbocation formed by a primary alcohol is unstable, and thus will not readily occur. This results in no observable reaction at room temperature. A tertiary alcohol will form a stable carbocation, resulting in a rapid reaction upon the addition of the Lucas reagent. This results in a second phase appearing in the reaction mixture because the final halogenated product is insoluble in water. Secondary alcohols will form a less stable carbocation than tertiary alcohols, but the reaction will occur at room temperature in a matter of minutes. Therefore, the Lucas Test can help differentiate between primary, secondary, and tertiary alcohols.</p>
<h4><strong>References</strong></h4>
<ol>
	<li style="font-size:17px">Streitwieser, A., Heathcock, C.H., Kosower, E.M. (1998).<em> Introduction to Organic Chemistry.</em> Upper Saddle River, NJ: Prentice Hall.</li>
</ol>

Identifying Alcohols: Ferric Chloride Test, Jones Test, and Lucas Test - Concept

알코올 식별

Alcohols
Alcohols are organic compounds that are amongst the most recognizable and familiar, as they have wide-ranging applications and uses in everyday ...

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JoVE Lab Manual

화학

Ideal Gas Law

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Put on a lab coat, splash-proof safety glasses or goggles, and appropriate gloves for the chemicals you will be handling.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">The day before the lab, place the Dumas tubes in an oven at 125 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Two hours before the lab, remove the tubes from the oven and place them on a heat-resistant surface to cool. Distribute the tubes to each workstation once they have reached room temperature.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Place a barometer in an easily accessible location.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Set a plastic 250-mL beaker next to each analytical balance, and ensure that a box of laboratory wipes is available for each balance.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Set up a Bunsen burner in a fume hood, and place a striker and a test tube holder with the burner.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Set out the following glassware and equipment at each workstation (we suggest students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;500-mL Erlenmeyer flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;500-mL B&#252;chner flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;400-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;100-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;3-prong clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Ring fixture with square of wire gauze</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Bunsen burner and fire striker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;18-in length of latex tubing</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;18-in length of silicone tubing</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Thermometer and thermometer clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Single-hole #4 rubber stopper</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation and Dispensing of Unknown Liquids<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Label one 50-mL glass bottle and one 20-mL glass syringe as &#39;deionized water&#39;.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Label three 50-mL brown glass bottles, three 5-mL glass syringes, and three 25-mL glass beakers as &#8216;1&#8217; through &#8216;3&#8217;, and set them in a reserved fume hood.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Place a watch glass and 20-mL volumetric pipette next to each numbered bottle along with a pipetter. Set four 22-gauge, 6-in needles next to each syringe.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Group the glassware by label in a reserved fume hood according to their chemical compound: 1,2-dichloroethane, trichloroethylene, and 2-bromopropane.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Transfer at least 20 mL of each organohalide into its assigned bottle using a volumetric pipette: 1,2-dichloroethane in bottle 1, trichloroethylene in bottle 2, and 2-bromopropane in bottle 3. Cap each bottle once the liquid has been added.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Label a fifth 50-mL bottle, 5-mL glass syringe, and 25-mL beaker as &#39;acetone&#39;, and set a 22-gauge, 6-in needle next to the syringe.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Add about 20 mL of acetone in the appropriately labeled bottle and cap it.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">When it is time to dispense the unknown liquids during the laboratory, pour at least 5 mL of an unknown into the matching beaker. Submerge the tip of the matching syringe in the unknown and slowly withdraw the plunger to fill the syringe to the 5-mL mark. Connect the needle and set down the syringe on a weight paper. Cover the beaker with a watch glass after filling.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Repeat this process for each organohalide.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Call up each student group one by one. Insert the needle of the syringe with their assigned unknown liquid into the long neck of the Dumas tube and dispense 0.5 mL. Withdraw the needle, taking care to avoid letting the liquid drip on the outside of the tube. The student group can now begin their trial.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Dispense the unknowns for the rest of the lab in this way, refilling the syringes as needed. At the end of the lab, follow the same procedure to dispense 0.5 to 1 mL of acetone into each tube for cleaning by students. The amount of acetone will depend on the amount of water left over in the tube.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">If students need to re-weigh their empty Dumas tube in the middle of the experiment, you will flame dry the tube so that they can obtain an accurate measurement.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Light a Bunsen burner and hold the Dumas tube with a test tube holder, and pass the tube through the flame until it appears dry. <strong>Note: </strong>Three of the organohalides are flammable, so keep the opening of the tube away from the flame.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Then, hold the tube away from the flame for 1 min to allow any remaining vapor to condense. If you see liquid at the bottom of the tube after that, pass the tube through the flame again until it appears dry. Once no liquid condenses in the tube after one minute out of the flame, return the tube to the student group so that they can measure the mass.</div>
		</li>
	</ul>
	</li>
</ol>

Ideal Gas Law: Derivation, Assumptions, and the Dumas Method - Prep

이상 기체 법칙

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation for 10 students ...

Lab Manual Sub Articles

Extraction

<p>Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Place labeled containers for halogenated waste and solid reagent waste in a satellite waste hood. Place a glass waste container nearby.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Label a 20-mL vial as &#8216;unknown mixture&#8217;.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Measure out 0.5 g of cellulose and add it to the labeled vial. Measure out 4.75 g each of benzoic acid and caffeine and add them to the vial. Stir the solids well with a glass rod.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Tightly cap the vial and place it by the balances. Set a box of 90-mm circular filter paper by the balances. Make sure that the area has enough weighing boats, laboratory wipes, and clean spatulas.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
		<table align="center" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Lab stand with ring fixture</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Hotplate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Vacuum pump (or house vacuum)</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Medium 3-prong clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;125-mL separatory funnel with a stopper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;125-mL Erlenmeyer flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;10-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;50-mL glass graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;50-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;3&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;600-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;250-mL filter flask</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;B&#252;chner funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;60-mL glass funnels</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;100-mm watch glasses</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Glass stirring rods</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;24/40 rubber adapters</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Pasteur pipette bulbs</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;2&#160;&#160;&#160;&#160;Small spatulas</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Package of pH paper</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px; ">&#160;1&#160;&#160;&#160;&#160;Roll of lab tape and pen</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Distribute boxes of lab wipes and Pasteur pipettes around the lab so that each workstation has them nearby.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Set out anhydrous magnesium sulfate, sodium bicarbonate, vacuum grease, and several spatulas on a common bench.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Shortly before the lab, place labeled bottles of dichloromethane and 1 M NaOH in a fume hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Put a bottle of 3 M HCl in a separate fume hood to avoid inadvertent contact between acids and bases.</div>
		</li>
	</ul>
	</li>
</ol>

Extraction: Liquid-Liquid and Acid-Base Extraction - Prep

추출

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

	Preparation of the LaboratoryExpand
	Here, we show the laboratory preparation ...

Redox Reactions

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Making Solid Copper<button class="expand">Expand</button>
	<p>In the first half of this lab, you will make solid copper. In this reaction, the solid copper is first oxidized by the strong oxidizing agent, nitric acid, to form water and the salt copper nitrate. The copper nitrate quickly dissociates into the ions, copper(2+) and nitrate, in water. The nitrate anions are reduced to NO<sub>2</sub> gas, which is confirmed by the presence of reddish-brown fumes. The presence of copper ions is indicated by a greenish-blue tint. Adding a base to the mixture changes the color to blue as the copper(II) hydroxide precipitate is formed. The copper(II) hydroxide is relatively unstable; thus, when heat is introduced, a decomposition reaction occurs. In this reaction, water is removed to produce copper oxide, which appears as a black precipitate.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before starting the experiment, put on personal protective equipment, including a lab coat, gloves, and chemical splash goggles.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Weigh between 0.45 and 0.55 g of copper shot and record the exact mass. This experiment must be conducted in the hood, so bring the copper shot back to your workstation.
		<h4><strong>Table 1. Determining percent yield</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" width="100%">
			<tbody>
				<tr>
					<td>Mass<sub>Cu initial </sub>(g)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass<sub>Cu final</sub> (g)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Percent yield</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/43/Table 1_Determining Percent Yield.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Label a 10-mL graduated cylinder as &#8216;8 M nitric acid&#8217; and measure 8 mL of the solution provided by your instructor. Cover the cylinder with a watch glass when transporting the solution back to your hood.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">To begin the experiment, place the copper in a 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Slowly add the 8 mL of 8 M nitric acid while stirring with a stirring rod. <strong>Note:</strong> If the copper does not fully dissolve, place the beaker on a hotplate set to 30 &#176;C for approximately 2 min, while continually stirring. Here, the solid copper is reacting with the nitric acid to form copper nitrate, water, and NO<sub>2</sub> gas. Do not overheat the solution or the nitric acid will boil off.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Once the copper is fully dissolved, remove the beaker from the hotplate and set the temperature on the hotplate to 85 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Measure 150 mL of deionized water using a 50-mL graduated cylinder and transfer the water into a 400-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Slowly, and while stirring, pour the copper solution into the 400-mL beaker containing water.<br />
		<strong>Note:</strong> If there is copper solution left in the beaker, use a pipette to rinse the rest of the solution into the larger beaker.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Label a 50-mL graduated cylinder with &#39;3 M NaOH&#8217; and obtain 20 - 30 mL of 3 M NaOH from your instructor. Cover the graduated cylinder with a watch glass while carefully carrying it back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Add 10 mL of the NaOH to the 400-mL beaker while stirring.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Test the pH of the solution using pH paper. Continue adding NaOH until the solution is basic or about pH 10. Here, the copper nitrate reacts with NaOH to form the blue precipitate, copper(II) hydroxide, and sodium nitrate.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Place the 400-mL beaker on the hotplate and use a glass thermometer to monitor the temperature. Once the solution reaches 85 &#176;C, let it remain there for 10 min. Stir slowly with the stirring rod, as this will evenly distribute heat while not disrupting crystal formation.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After 10 min, turn off the hotplate and remove the beaker. Allow the beaker to cool to room temperature. As the solution cools, a dark precipitate will appear at the bottom of the beaker, while a clear and colorless liquid, the supernatant, will separate towards the top. The crystals are the copper oxide.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the solution has cooled completely, label a new beaker as &#39;aqueous waste&#39;, and remove the supernatant by carefully pouring it into the waste container. Try not to lose the precipitate at the bottom of the beaker.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Set up the filtration system by attaching the silicone tubing between the house vacuum and the vacuum flask. Place a B&#252;chner funnel into the vacuum flask using a rubber adapter. Now, place one filter paper in the B&#252;chner funnel and wet it with deionized water.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Turn on the vacuum. Pour the precipitate into the B&#252;chner funnel and direct it into the center with the stirring rod. Use deionized water to rinse all the precipitate into the funnel. When all the precipitate is out of the beaker, turn off the vacuum.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Remove the filter paper with the precipitate and place it into the beaker. The dark precipitant on the filter paper is the copper oxide, which should be left to dry for a day or two before it is used in the next reaction.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Clean up from the experiment. First, empty the liquid in the filter flask into the aqueous waste beaker. Then, test the aqueous waste with pH paper. <strong>Note: </strong>Any waste that is higher than pH 10 should be neutralized with citric acid. Any waste below pH 4 should be neutralized with baking soda.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Neutralize the unused NaOH with citric acid and then pour it down the drain with copious amounts of water. Anything containing black particles indicates the presence of copper oxide and should be disposed of in the heavy metal drum container.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Making Copper Oxide<button class="expand">Expand</button>
	<p>The copper oxide produced in the previous lab section is insoluble in water but can be dissolved in sulfuric acid. When this occurs, the copper(2+) cations and the sulfate anions are reintroduced through a double displacement reaction. When zinc is added in the final step, the copper(2+) ions are reduced to solid copper through a single displacement reaction.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-2">
		<div class="lab-step">Put on the appropriate protective equipment, such as a lab coat, gloves, and chemical splash goggles.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Obtain the dried copper oxide that was prepared in the last lab exercise and remove as much copper oxide from the filter paper as possible using a rubber policeman. Place the solid and filter paper back into the same 400-mL beaker it was stored in.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Label a 10-mL graduated cylinder as &#39;5.93 M sulfuric acid&#39; and obtain 8 mL of sulfuric acid from the instructor. Cover the graduated cylinder with a watch glass, and carefully carry the sulfuric acid to your hood. <strong>Note: </strong>Perform the next step of the experiment in the hood.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use a plastic pipette to add about 5 mL of sulfuric acid directly onto the filter paper in the beaker.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Then, hold the filter paper with tweezers and use a metal spatula to scrape off as much solid as possible without ripping the filter paper. Use as little sulfuric acid as possible to rinse the copper oxide, as excess sulfuric acid will require more zinc later.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Rinse copper oxide from the filter paper with 2 mL of deionized water and add a little more deionized water to the beaker. Once the copper oxide has been removed from the filter paper, dispose of the filter paper as solid waste.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Stir the solution until the copper oxide is fully dissolved. If the solid does not dissolve, place the beaker on the hot plate and heat it to about 60 &#176;C.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Add 3 - 5 mL of water and stir to dissolve. Be careful not to go higher than this temperature, as the liquid could evaporate leaving a precipitate of copper sulfate.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Weigh 1.2 g of zinc and record the exact mass in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Slowly add half of the zinc to the solution. Crush and stir the zinc using the stirring rod to fully dissolve all particles. The solution should be colorless. If color is still present, add more zinc until the color disappears.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Once the color fully disappears, weigh the remaining zinc in the weigh pan, and subtract it from the original mass to obtain the mass of the zinc used. Here, the zinc has reacted with the copper sulfate to form zinc sulfate and solid copper.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Decant the supernatant from your solution into the container labeled &#39;aqueous waste&#39;. Then, preheat the hot plate to 40 &#176;C. Obtain 10 mL of 6 M hydrochloric acid from your instructor in a labeled graduated cylinder. Cover the opening of the cylinder with a watch glass and bring the acid back to your hood.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Add 5 mL of the hydrochloric acid to the remaining precipitate to dissolve any zinc present within the solid copper.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Place the beaker on the preheated hot plate and warm the solution for 2 min. The bubbling observed here is the neutralization reaction taking place to dissolve the zinc. Make sure that the reaction does not boil. Then, turn the heat setting on the hot plate off.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Once the bubbling subsides, add 2 - 3 mL of additional hydrochloric acid. Continue adding acid in 1-mL increments until the bubbling completely stops.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Decant the supernatant, wash the precipitate with 5 - 10 mL of water, and decant any remaining supernatant. <strong>Note: </strong>If the copper particles are too small to decant the liquid without losing them, set up a B&#252;chner funnel apparatus with filter paper and no vacuum. Pour the solution through the funnel so the particles are retained on the filter paper, then return the particles to the beaker. Repeat the wash and decant steps two more times.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Replace the filter paper in the B&#252;chner funnel with a fresh one and wet it slightly with water. Then, turn on the vacuum for a moment to help the filter settle into place.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Add approximately 5 mL of water to the copper precipitate, and then pour it into the funnel in the center of the filter paper. Stir the copper gently to remove the impurities while taking care not to rip the filter paper.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Turn the vacuum on, and let it run for 2 min.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Close the vacuum and disconnect the tubing from the flask. Remove the funnel and dispose of the liquid in the aqueous waste container.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Reconnect the B&#252;chner funnel setup and reattach the vacuum tubing.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Obtain about 10 mL of methanol and pour 3 mL onto the copper in the funnel. Then, turn on the vacuum and let the copper dry for 2 min.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Dry the precipitate with methanol two more times, running the vacuum after each wash. During the last drying phase, place a large rubber stopper on the B&#252;chner funnel, and let the vacuum run for 5 min. Then, close the vacuum and disconnect the vacuum tube.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Remove the large stopper and let the copper air dry for 30 min.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">Label an empty beaker as &#39;organic waste&#39; and pour the liquid from the filter flask into the organic waste container.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Tare an empty weighing pan and transfer as much of the copper as possible from the filter paper to it. Weigh the copper and record its mass.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Clean up from the lab exercise. Dispose of the organic waste and excess methanol in the organic waste container. Test the aqueous waste with pH paper and neutralize it with either citric acid for highly basic pH or baking soda for acidic pH.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Dispose of the supernatant liquid in the heavy metal drum waste container because it contains zinc. The unused sulfuric acid and hydrochloric acid should be neutralized with baking soda and then washed with plenty of water down the sink.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Determine if the product is pure. Spread some of the product on an indicating desiccant to test for the presence of methanol. The desiccant will change from blue to turquoise if methanol is present.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Drop a few drops of 6 M HCl onto the product to see if it will react with any NaOH that may be left over. If NaOH is present, you will see bubbling.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">If the product is copper, it should look like dark-red granules. If the product is black, you have failed to make copper and instead made copper oxide.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Calculate the percent yield using the initial and final mass of copper. The percent yield is the ratio of the actual yield, in this case, the final mass of copper, to the theoretical yield, or the initial mass of the copper, multiplied by 100. It is impossible to obtain a percent yield greater than 100. If this occurs, there was an error in calculation &#8212; or the product was still wet &#8212; which added more weight.</div>
		</li>
	</ul>
	</li>
</ol>

Redox Reaction: Oxidation States - Procedure

산화 환원 반응

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Making Solid CopperExpand
	In the first half of this lab, you will make solid copper. In this ...

Metal Flame Emission

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Metal Flame Emission Testing<button class="expand">Expand</button>
	<p>In this lab, you will excite the electrons of six metal chloride salts with heat from a flame and measure the wavelength of light emitted by the relaxing electrons. The emitted light is in the visible spectrum, so you will see different colored flames for the different metals.</p>
	<p>At the end of the lab, you will measure the emission spectrum of a sparkler and use the data you collected from the metal salts to identify the metals in the sparkler. To help with this process, make a table in your lab notebook to record the wavelengths emitted from ambient light, the salts, and the sparkler.</p>
	<h4><strong>Table 1: Observed Wavelengths</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Run #</strong></td>
				<td style="text-align: center;"><strong>Source</strong></td>
				<td style="text-align: center;"><strong>Recorded wavelength(s)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">Room lights</td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">NaCl</td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">LiCl</td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">KCl</td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">SrCl<sub>2</sub></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">BaCl<sub>2</sub></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">CuCl<sub>2</sub></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">Sparkler</td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/48/Table_1._Observed_Wavelengths.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>During this lab, you will hold a salt-coated applicator at the edge of a Bunsen burner flame to excite the metal salts. If the applicator catches fire, immediately quench it in a beaker of water. For excitation, you will hold a detector near the flame and the sparkler to measure the wavelengths of the emitted light. Keep a safe distance to protect yourself and the detector. As some tasks are performed simultaneously, we suggest that you work in groups of four with each student completing tasks as needed.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-4">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Set up the Bunsen burner in the fume hood.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Fill a 400-mL beaker with tap water and bring it to the hood. Place the salts, applicators, and tap water within easy reach on one side of the Bunsen burner.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Turn on the hand-held spectrophotometer and familiarize yourself with its components.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Create a new experiment and configure the spectrometer to measure emission intensity. Set the sample time to 200 ms and the number of readings at a given wavelength to 1. Record the settings and the experiment name in your lab notebooks.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the spectrum of the room lights. Have one or two students hold the optical detector as close to the lights as possible without disconnecting the cables. Once ready, have a different student start the spectrometer. The spectrum should be clear, with minimal noise and no off-scale peaks. <strong>Note: </strong>If you have trouble, hold the probe in a nearly closed drawer or box to shield it from sunlight while pointing it at the room lights.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Immediately capture the spectrum when it looks good. Confirm that you have a high-quality spectrum before saving it. Record the wavelengths of the peaks in your data table.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure the emission spectra of the metal salts. Ignite the Bunsen burner and adjust the collar until the flame distinctly separates into an inner and outer flame.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Bring the spectrometer to the hood and arrange the cables so that the detector can be held about one inch from the flame.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Have one student prepare the spectrometer for the run. This student should have a clear view of the burner so they can begin acquiring data as soon as the salt enters the flame. Then, have another student hold the detector no closer than one inch from the flame. <strong>Note: </strong>Any closer risks damaging the detector. But you won&#39;t get a clear spectrum if the detector is too far away.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Have a different student take one of the applicators from the beaker of water and dip it into the tube of NaCl to coat the wet cotton with the salt. Then, carefully insert the salt-coated applicator about 1 mm into the outer flame, which will change color.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Start the spectrometer when the applicator enters the flame.<strong> Note: </strong>If the peak intensities are off-scale, adjust the detector position until all peaks are within detection limits. If the flame color change is hard to see, pick up more salt on the same applicator and try again until you see an intense color. If the spectrum is noisy or weak, try pointing the detector at a different part of the flame.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">When you see a good spectrum, capture and save it. Everyone should review the spectrum to ensure that it is of good quality. Write down the emission wavelengths in your table.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Submerge the NaCl applicator in your wastewater beaker. Always use a different applicator for each salt.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Follow the same procedure to test the remaining five salts, being careful to avoid cross-contamination between the salts. Once you have tested the last salt and recorded the wavelengths, set up data collection for the sparkler.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">At the instructor&#39;s hood, light the sparkler with the Bunsen burner and place it handle down in the Erlenmeyer flask. Remember, the sparks may be very hot. Turn off the gas to extinguish the Bunsen burner.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">As one student holds the detector close to the sparkler, have another student start data collection. Position the detector to get as many peaks in scale as possible without losing the less intense peaks.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Capture and save the spectrum once it looks good. Remember to write down the wavelengths in your table. <strong>Note: </strong>If the sparkler is long-lived, collect a second spectrum and either choose the best one or keep them both for the analysis.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Let the sparkler burn out in the hood while you send the collected data to everyone in the group and shut down the data acquisition system.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Bring the copper, barium, and strontium tubes to the waste hood and thoroughly rinse the tubes with deionized water into the designated waste container.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Flush the sodium, potassium, and lithium salts down the drain and rinse the remaining test tubes. Put away the test tubes and the other equipment.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Once the ashes and sparkler handle have cooled, dump them in the trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, plot the data for each run with intensity on the y-axis and wavelength on the x-axis.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Compare the sparkler spectrum to the background spectrum from the room lights. Identify which small peaks may be from the room lights.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Compare the metal spectra to the sparkler spectrum. Sodium appears to be the source of the peak at about 589 nm. There are no peaks matching the lithium spectrum, but potassium matches the large peak at 770 nm.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">The sparkler spectrum doesn&#39;t seem to have strontium peaks. However, barium is a good match for several peaks in the sparkler spectrum. Lastly, no copper seems to be present in the sparkler. Thus, we conclude that the sparkler was burning sodium, potassium, and barium when the spectrum was acquired.</div>
		</li>
	</ul>
	</li>
</ol>

금속 화염 방출

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Metal Flame Emission TestingExpand
	In this lab, you will excite the electrons of six metal ...

Basic Organic Chemistry Techniques

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Synthesis of Benzoic Acid<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll learn several important techniques that you&#39;ll use throughout this class. This includes measuring the mass or volume of reactants and products, methods of heating and cooling reaction mixtures, proper selection and handling of glassware, and separating solids and liquids by vacuum filtration. First, you&#39;ll synthesize benzoic acid by protonating sodium benzoate with hydrochloric acid, or HCl, using water as the solvent. Sodium benzoate is very soluble in water, but benzoic acid is not, so you can filter the solid product out of the solution.</p>
	<p>You&#39;ll use vacuum filtration, which is used for powdery solids, to filter out the benzoic acid. Benzoic acid is less soluble in water at lower temperatures, so you&#39;ll collect more product if you cool the reaction solution before you filter it. When you do this lab, remember that HCl is toxic and a strong acid. It&#39;s always best to work with strong acids and organic solvents in a fume hood. If you spill any HCl, follow your lab&#39;s procedures for neutralizing and cleaning up strong acids.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves. Change your gloves if you get acid or organic solvent on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Bring a clean 50-mL beaker and your lab notebook to the balance, and then tare a clean empty weighing boat on the balance.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use a clean spatula to measure out ~2 g of sodium benzoate.<strong> Note:</strong> Stay between 1.9 and 2.1 g.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Dispose of any extra sodium benzoate into the appropriate waste container. Close the bottle of sodium benzoate when you&#39;re done with it.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Write down the mass of sodium benzoate and pour it into your beaker. Clean the spatula with a lab wipe so that it can be used again and return to your fume hood.
		<h4><strong>Table 1: Reaction yield of benzoic acid</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Mass of sodium<br />
					benzoate (g)</strong></td>
					<td style="text-align: center;"><strong>Moles of sodium<br />
					benzoate</strong></td>
					<td style="text-align: center;"><strong>Volume of<br />
					HCl (mL)</strong></td>
					<td style="text-align: center;"><strong>Moles of<br />
					HCl</strong></td>
					<td style="text-align: center;"><strong>Moles of benzoic<br />
					acid</strong></td>
					<td style="text-align: center;"><strong>Mass of benzoic<br />
					acid (g)</strong></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td><strong>Theoretical yield</strong></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td style="text-align: center;">-</td>
					<td><strong>Percent yield</strong></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/56/Table_1._Reaction_yield_of_benzoic_acid.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Get a clean 10-mL graduated cylinder and measure 10 mL of deionized water. Pour the water into the beaker of sodium benzoate. Stir the solution with a glass rod until the sodium benzoate has completely dissolved.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Obtain a clean 5-mL graduated cylinder and measure 5 mL of 3 M HCl. <strong>Note: </strong>If you pour too much, use a Pasteur pipette to transfer the excess to a small beaker labeled &#8216;acid waste&#8217;. Close the bottle and put it back when you&#39;re done.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a clean Pasteur pipette to draw up a small amount of HCl from your graduated cylinder. Start adding the HCl to the sodium benzoate solution at a rate of about one drop every 6 seconds or 10 drops/min. Stir the solution frequently with the glass rod. This should take about 10 min. <strong>Note:</strong> As you add more HCl, you&#39;ll start to see benzoic acid precipitating from the solution.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">When you are done, fill a 600-mL beaker about halfway with crushed ice and add tap water to just fill the spaces between the ice to make an ice bath.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Place the ice bath by a lab stand in your fume hood and use a medium clamp to hold the reaction beaker in the bath. Make sure that the surface of the solution is below the ice.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Clamp a thermometer in the reaction solution.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">While the reaction cools, obtain 25 mL of deionized water in a 50-mL graduated cylinder. Place the graduated cylinder in a 600-mL beaker and pack ice around it to cool the water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Once the reaction solution reaches 10 &#176;C, it&#39;s ready to be filtered. Set up another lab stand and obtain a clean 250-mL filter flask. Clamp the flask upright on the lab stand.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Ensure that your vacuum tubing is connected to the vacuum source, and then carefully push the free end of the tubing onto the barbed arm of the flask.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Place a rubber adapter and a B&#252;chner funnel in the mouth of the flask. Confirm that the reaction solution has reached 10 &#176;C and remove the thermometer from the beaker.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Obtain a piece of circular filter paper and place it in the B&#252;chner funnel so that the small holes are completely covered.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Get a new Pasteur pipette, and use a few drops of cold deionized water to wet the filter paper. <strong>Note: </strong>This helps keep the filter paper in place when you pour the solution into the funnel.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Open the vacuum line part way to reduce the pressure in the flask.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Remove the beaker from the ice bath, stir the solution well with the glass rod until the solid is swirling around in the liquid, and then slowly pour it onto the filter paper. <strong>Note:</strong> If the liquid does not move quickly through the funnel, make sure that there are no gaps between the funnel, adapter, and flask, and then gradually open the vacuum until the liquid rapidly flows into the flask.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Use a clean Pasteur pipette to rinse the inner walls of the beaker with ~10 mL of cold deionized water and pour the rinse into the funnel. Pour the rest of the cold deionized water over the benzoic acid on the filter to wash it. <strong>Note:</strong> This is an important step that removes impurities, side products, and unreacted compounds.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Leave the benzoic acid in the funnel with the vacuum running until it is completely dry. <strong>Note: </strong>This usually takes ~2 h, so let&#39;s continue to the second half of the lab in the meantime.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Refluxing Tetrahydrofuran (THF) and Measuring Product Mass<button class="expand">Expand</button>
	<p>While your benzoic acid dries, you&#39;ll practice refluxing tetrahydrofuran, or THF, which has a boiling point of 66 &#176;C. THF is volatile, flammable, and an irritant, so avoid letting it touch your skin and always work with it in a fume hood. Refluxing is a technique that constantly condenses the vapor and returns it to the flask. This allows reactions to be performed at high temperatures, such as at the solvent&#39;s boiling point, without losing the solvent to evaporation.</p>
	<p>Refluxing is achieved with a special piece of glassware called a condenser, which is attached directly to your flask. The condenser is cooled with water flowing through the outer chamber. As vapor rises through the condenser, it loses heat to the cool walls, condenses, and drips back into the flask.</p>
	<p>Refluxing is usually performed in a round-bottom flask. These flasks must be heated in a heating bath or with specialized equipment, but they provide more even heating than an Erlenmeyer flask or beaker as a result. Water baths are typically used for experiments performed below 80 &#8211; 100 &#176;C.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-4">
		<div class="lab-step">Set up the glassware and equipment for reflux. Place a lab jack by your available lab stand and set a stirring hotplate on top of it. Clamp a 50-mL round-bottom flask at least 12 cm above the hotplate.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring a 50-mL graduated cylinder to the solvent hood and measure 25 mL of THF. If you pour too much, label a small beaker &#8216;organic waste&#8217; and pipette the excess into that. Remember to cap the bottle when you&#39;re done.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Carefully pour the THF into the round-bottom flask, clean up any spills with a lab wipe or paper towel, and add a small oblong stir bar to the flask.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Position a medium clamp ~20 cm above the flask. Obtain a condenser and apply vacuum grease to the lower ground-glass joint.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Fit the condenser into the flask and rotate them in opposite directions until the vacuum grease is evenly spread between the two ground-glass surfaces.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Clamp the condenser in place and use a lab wipe to remove excess grease from the connection between the two pieces of glassware.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Attach rubber tubing to each of the ports on the condenser. Place the free end of the tubing connected to the top port, which is the outlet, in the drain. Connect the free end of the tubing from the bottom port, which is the inlet, to the waterline.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Fill a 250-mL beaker with ~200 mL of tap water and place this water bath on the hotplate. Use the lab jack to raise the bath until the water level is about 2/3 of the way up the spherical part of the flask. This will ensure the THF heats evenly.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Secure a thermometer (range 0 &#8211; 100 &#176;C) in the bath using the thermometer clamp.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Slowly open the water tap, watch the condenser for leaks as it fills, and confirm that the water is flowing into the drain.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Slowly turn up the magnetic stirrer until the THF is stirring well. If the stir bar bounces around, turn down the stir motor and try again more slowly.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Start heating the water bath to about 70 &#176;C until the THF starts to boil. <strong>Note: </strong>You&#39;ll see vapor condensing in the lower part of the condenser).</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Monitor the refluxing THF for 10 min. <strong>Note:</strong> Turn down the heat if the bath goes above 70 &#176;C or if vapor leaves the condenser.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">After 10 min, remove the thermometer and turn off the heat and the stir motor. Carefully lower the lab jack until the flask is clear of the water bath. Wait at least 10 more min for the setup to cool, then unplug and remove the hotplate.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Turn off the water tap, unclamp the condenser, and carefully detach it from the flask. Tilt the condenser until most of the water has flowed through the outlet tubing into the drain. Then, disconnect the inlet tubing from the condenser and let the remaining water flow into the drain. Do the same for the outlet tubing.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Use lab wipes to remove the vacuum grease from the condenser and the flask, then retrieve the stir bar from the flask.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Bring the flask to the waste hood and empty it into the non-halogenated organic waste container. Pour any excess HCl or THF into the acid and organic wastes, respectively. Empty the ice and water baths into the sink and rinse the beakers with tap water.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Clean most of your other glassware while the benzoic acid from earlier finishes drying according to your lab&#8217;s standard practices. Usually, you&#39;ll rinse your glassware both with an organic solvent, like acetone, and with detergent and deionized water. <strong>Note:</strong> Your lab may have specific procedures for glassware used with acids. If so, follow that procedure to clean the graduated cylinder used with HCl and your acid waste beaker, if applicable.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Remove the reusable pipette bulbs and dispose of your pipettes in the glass waste container. Throw out used lab wipes and paper towels in the lab trash.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Check on your benzoic acid. If it is dry, turn off the vacuum and break the vacuum seal by disconnecting the tubing from the filter flask. Gently place the funnel in it 250-mL beaker and bring it and your lab notebook to the balance.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Tare a weighing boat and use a clean spatula to transfer the product to the weighing boat. Record the mass of your product in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Then, put the product in the lab trash and wipe off the spatula. Dispose of the filtrate in the aqueous waste container.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">Clean the rest of your glassware, throw out the filter paper and other waste in the lab trash, and put away your lab equipment. Lastly, clean the floor of your fume hood with a damp paper towel.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the reaction yield for the protonation of sodium benzoate. Identify the reagent that the reaction runs out of first, which is called the limiting reagent.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Calculate the number of moles of sodium benzoate and HCl that you used. <strong>Note:</strong> You used about 0.014 moles of sodium benzoate and 0.015 moles of HCl. Thus, the maximum yield of benzoic acid is equal to the starting amount of sodium benzoate in moles.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Convert the mass of your benzoic acid to moles, divide that by the moles of sodium benzoate that you used, and multiply it by 100 to get the percent yield. <strong>Note:</strong> The recovered yield for this reaction is usually between 80% and 98% because a small amount of benzoic acid stays in solution. If the yield is above 100%, then it&#39;s likely that your product was still wet when you weighed it. Impurities, unreacted compounds, and products of side reactions can also affect the accuracy of yield calculations.</div>
		</li>
	</ul>
	</li>
</ol>

기본 유기 화학 기법

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Synthesis of Benzoic AcidExpand
	In this lab, you&#39;ll ...

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse=""><strong>Preparing 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7</strong><button class="expand">Expand</button>
	<p>In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.</p>
	<p>Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before starting the experiment, calculate the mass of monosodium phosphate that you will need to make 200 mL of a 50 mM solution.
		<h4><strong>Table 1: Preparation of 50 mM NaH<sub>2</sub>PO<sub>4</sub> Buffer, pH 7.0</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" height="247" width="358">
			<tbody>
				<tr>
					<td colspan="2"><strong>Molar mass of NaH<sub>2</sub>PO<sub>4</sub> = 119.98 g/mol</strong></td>
				</tr>
				<tr>
					<td>Solution volume (mL)</td>
					<td style="text-align: center;">200</td>
				</tr>
				<tr>
					<td>Molarity (mM)</td>
					<td style="text-align: center;">50</td>
				</tr>
				<tr>
					<td>Moles of NaH<sub>2</sub>PO<sub>4</sub> (mol)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Mass needed (mg)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Initial volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Final volume of 1 M NaOH (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of NaOH used (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td>Volume of DI water needed (mL)</td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_1._Preparation_of_50_mM_NaH2PO4_Buffer,_pH_7.0.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">To begin, put on the necessary personal protective equipment, including a lab coat, splash-resistant safety glasses, and gloves. <strong>Note: </strong>This section of the lab uses NaOH, which is corrosive and toxic. Use caution when pouring and transporting NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Fill a 250-mL plastic wash bottle with deionized water.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Label two 100-mL beakers, one for neutral aqueous waste and one for basic aqueous waste.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Tare weighing paper, and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Record the exact amount of monosodium phosphate in your lab notebook. <strong>Note: </strong>Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement and close the container tightly when you are done with it.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Place the monosodium phosphate in a 400-mL beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use a graduated cylinder to measure out 175 mL of deionized water. Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Stir the solution until the salt has dissolved completely and the solution appears homogeneous. This usually takes 2 - 3 min.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Measure 15 mL of deionized water and pour it into the beaker. Continue stirring the solution until it appears homogeneous again (about 1 &#8211; 2 min). Turn off the stir motor.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Bring a 10-mL graduated cylinder and watch glass to the dispensing hood and measure out 10 mL of 1 M NaOH. Note the exact volume in the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Cover your NaOH with the watch glass, and carefully bring it to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add NaOH to the stirring solution in a drop-wise manner. Once the buffer pH reaches 7.0, return any NaOH still in the pipette to the graduated cylinder.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Calculate the volume of NaOH that you added to the buffer. Subtract that volume from 10 mL to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 mL.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Measure the deionized water with another 10-mL graduated cylinder and add it to the stirring solution to finish making the buffer.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Label a 250-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer, pH 7.0&#39;. Retrieve the magnetic stir bar from the solution.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Use a funnel to pour the buffer solution into the labeled bottle and cap it tightly.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse=""><strong>Absorption Spectroscopy of Free and Bound Neutral Red</strong><button class="expand">Expand</button>
	<p>Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.</p>
	<p>After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Draw the following table in your lab notebook. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use. The 10th cuvette will be a deionized water blank. <strong> Note: </strong>Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
		<h4><strong>Table 2: Absorbance of Free and Bound Neutral Red</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Cuvette #</strong></td>
					<td style="text-align: center;"><strong>Buffer pH </strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max </sub>(Free NRH<sup>+</sup>)</strong></td>
					<td style="text-align: center;"><strong>Abs. at &#955;<sub>max</sub> (Bound NRH)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1</td>
					<td style="text-align: center;">5.0</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">2</td>
					<td style="text-align: center;">5.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">6.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">6.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">7.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">6</td>
					<td style="text-align: center;">7.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">7</td>
					<td style="text-align: center;">8.0</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">8</td>
					<td style="text-align: center;">8.5</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">9</td>
					<td style="text-align: center;">11</td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">10</td>
					<td style="text-align: center;">blank</td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/45/Table_2._Absorbance_of_Free_and_Bound_Neutral_Red.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Obtain ten 1.5-mL cuvettes and caps and label the caps 1 through 10 to match the table in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 25-mL beaker as &#8216;DIH2O&#8217; and fill it with deionized water.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Flush your neutral aqueous waste down the drain and relabel the beaker as &#39;aqueous buffer waste&#39;.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Label a 400-mL beaker for used micropipette tips.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Attach a tip to a 1-mL micropipette and use it to dispense 1000 &#181;L of deionized water into cuvette 10. This is your solvent blank.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Place 925 &#181;L of your pH 7.0 monosodium phosphate buffer in cuvette 5.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 &#181;L of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once you have finished, bring the buffers back to your workbench. There, set a 200-&#181;L micropipette to dispense 75 &#181;L, and attach a tip to the micropipette.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Dispense 75 &#181;L of neutral red into each of the nine buffer cuvettes. Invert each cuvette several times to thoroughly mix the solutions. <strong>Note: </strong>Replace the pipette tip if it touches a buffer.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Turn on a hand-held spectrophotometer and allow the light source to warm up. Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Insert the cuvette of deionized water and acquire a spectrum of the deionized water. Set it as a solvent background or blank.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Then, remove the blank and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum. Record this wavelength in your lab notebook as lambda max for protonated free neutral red.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Save the data and remove the cuvette. Record the absorbance in your notebook, then collect spectra for cuvettes 2 - 9 using the same procedure.</div>
		</li>
		<li data-sub-note-item="2-19">
		<div class="lab-step">Record the wavelength of the isosbestic point in your lab notebook. That is where all the spectra cross at a single wavelength. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.</div>
		</li>
		<li data-sub-note-item="2-20">
		<div class="lab-step">Once you finish collecting spectra for all nine cuvettes, save and export the data.</div>
		</li>
		<li data-sub-note-item="2-21">
		<div class="lab-step">Create a new experiment to measure absorbance versus wavelength for the bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-22">
		<div class="lab-step">Fit a new tip to the 200-&#181;L micropipette and add 75 &#181;L of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions.</div>
		</li>
		<li data-sub-note-item="2-23">
		<div class="lab-step">Insert cuvette 10 into the spectrophotometer and set it as the solvent blank.</div>
		</li>
		<li data-sub-note-item="2-24">
		<div class="lab-step">Acquire a spectrum of the lowest pH sample, and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red.</div>
		</li>
		<li data-sub-note-item="2-25">
		<div class="lab-step">Acquire spectra for cuvettes 2 - 9 in the same way as you did for the free neutral red samples. Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point.</div>
		</li>
		<li data-sub-note-item="2-26">
		<div class="lab-step">When you are done, save your data, export it for later analysis, and shut down the spectrophotometer.</div>
		</li>
		<li data-sub-note-item="2-27">
		<div class="lab-step">Put away all equipment and dispose of your used pipette tips in an approved waste container or trash bin.</div>
		</li>
		<li data-sub-note-item="2-28">
		<div class="lab-step">Empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="2-29">
		<div class="lab-step">Empty the excess NaOH into the basic waste and rinse the graduated cylinder with water.</div>
		</li>
		<li data-sub-note-item="2-30">
		<div class="lab-step">Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste.</div>
		</li>
		<li data-sub-note-item="2-31">
		<div class="lab-step">Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse=""><strong>Results</strong><button class="expand">Expand</button>
	<p>Now, let&#39;s analyze our absorbance data to determine the pKa&#39;s of neutral red.</p>
	<h4><strong>Table 3: Determination of Neutral Red pKa&#39;s</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td></td>
				<td>Free NRH<sup>+</sup></td>
				<td>Bound NRH<sup>+</sup></td>
			</tr>
			<tr>
				<td>&#160;&#955;<sub>max</sub> (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;&#916;A (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;Midpoint (nm)</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td>&#160;pKa</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/45/Table_3._Determination_of_Neutral_Red_pKa.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Plot both sets of intensity data with absorbance intensity at lambda max on the y-axis and pH on the x-axis, with the points joined by smooth lines.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the difference between the starting and ending absorbance intensities for each data series.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Determine the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Find where the midpoint occurs on each line and identify the pH value at that point. These pH values are the pKa&#39;s of free and bound neutral red.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Here, we see an increase in pKa of about one between free and bound neutral red, indicating that bound-protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Procedure

버퍼

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparing 50 mM NaH2PO4 Buffer, pH 7Expand
	In the first part of this experiment, you will ...

Thin-Layer Chromatography

<p>Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Effects of Mobile Phase Composition on Compound Separation<button class="expand">Expand</button>
	<p>In this section of the lab, you will use TLC to compare how well pure hexane and a mixture of 60% hexane and 40% ethyl acetate can separate phenol, benzoic acid, and butyl phenyl ether by measuring the distance the compound traveled in each solvent.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before starting the lab, put on a lab coat, safety glasses, and nitrile gloves. Note: Hexane and ethyl acetate are both volatile, toxic, and flammable, so always work with them in a fume hood. Change your gloves if you get solvent on them, as hexane and ethyl acetate will permeate nitrile gloves within minutes.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Now, obtain a paper towel and lay it on the benchtop. Carefully pick up a TLC plate using tweezers at the very end of the plate.<strong> Note: </strong>Always hold TLC plates by the edges or at the very top with tweezers to avoid damaging or contaminating the silica gel.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Look for chips or scratches in the silica gel and get a new plate if necessary. It&#39;s fine if there is a little chipping at only one end, since that can be the top.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Place the plate on the paper towel in your fume hood, and lay a metric ruler next to it. Use a graphite pencil to gently make a small mark on the silica gel ~1 cm from the top of the plate. Write &#8216;100&#8217; on the plate above the mark.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, gently mark the plate 1 cm above the bottom on both sides. Carefully trace a straight line between the bottom marks to make the starting line.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, add 3 evenly spaced small marks to the starting line with the outer marks at least 0.5 cm from the sides of the plate. <strong>Note:</strong> It&#39;s important for the silica gel to be undamaged here, so if you scratch it with the pencil, prepare a new plate.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, trace the outline of the TLC plate in your lab notebook and precisely copy the marks on the plate. Label the three marks as &#8216;phenol&#8217;, &#8216;benzoic acid&#8217;, and &#8216;butyl phenyl ether&#8217;. You may also want to label the marks on the plate itself.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare a second TLC plate labeled &#8216;60/40&#8217; in the same way. Now, bring the labeled plates to the common bench and locate the 1 mg/mL solutions of phenol, benzoic acid, and butyl phenyl ether in ethanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Dip a clean capillary into one of the three solutions. Briefly and lightly dab the capillary squarely against the intersection between the starting line and the mark for that compound. You should spot twice for each compound.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use the same capillary to spot the same solution on your second TLC plate. The spots should be no more than 2 mm in diameter. Then, discard the capillary and close the vial.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Spot the other two solutions on the plates, in the same way, using clean capillaries. Be careful not to cross-contaminate the solutions. <strong>Note</strong>: If a spot is very light or very small, wait for it to dry, and then spot the same solution on top of the dry spot. If the spot is still wet, capillary action could make it too wide and dilute.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have made three spots on each plate, discard the capillaries and bring the plates back to your hood. Keep the plates lying flat while they dry.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, label a 50-mL jar &#8216;100% hexane&#8217; and bring the jar, its cap, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Measure 2 &#8211; 3 mL of pure hexane with your graduated cylinder and pour it into the jar to fill it to a depth of 0.62 &#8211; 0.7 cm. Cap the bottle and jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a second 50-mL jar &#8216;60% hexane, 40% ethyl acetate&#8217; and bring it and a clean graduated cylinder to the solvent hood. Measure 2 &#8211; 3 mL of the 60/40 mixture into the jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Stand the plates next to the jars to compare the relative position of the solvent and the spots. The solvent level should be below the spots for each plate.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Then, obtain three pieces of filter paper, put a filter paper upright against the wall in each jar, and then cap the jars tightly.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Initial each plate above the mark so you can identify them later. Confirm that the filter papers in the jars have become saturated with solvent and that the spots on the plates are completely dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, open the 100% hexane jar and use tweezers to gently place the 100 plate in the jar, keeping the plate level with the solvent surface as it enters the solvent. Close the jar tightly, being careful not to jostle the plate.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Leave the jar still as the solvent travels up the plate. Development in pure hexane usually takes about 7 &#8211; 10 min. Keep an eye on the plate to make sure that the solvent doesn&#39;t reach the top.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">When the pure hexane solvent front is about 1 cm from the top of the plate, retrieve it using tweezers. Lay it on the paper towel with the silica gel facing up and quickly mark the solvent front with a line and pencil across the plate. Leave the plate lying flat as the solvent evaporates.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Place the other plate in the 60/40 eluent in the same way. Wait until the solvent front of the 60/40 plate is about 1 cm from the top, and then retrieve it and quickly mark the solvent front in the same way.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Once the 100% hexane plate is completely dry, pick it up with tweezers and bring it and a pencil to the UV lamp on the common bench.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Place the plate under the lamp and illuminate it with UV light. The plate will glow green because of a fluorescent marker in the silica gel layer. The dark spots are the compounds.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Carefully outline every spot in pencil, even if they haven&#39;t moved very far.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, turn off the lamp and bring your plate to the iodine development hood. Open the iodine chamber. Use tweezers to gently place the plate upright in the iodine chamber, and then cover the chamber again.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Wait 3 &#8211; 4 min for the iodine vapor to stay in the spots. Then, retrieve the plate with tweezers, close the chamber, and outline the brown and yellow spots on the plate. These spots will fade, so mark them quickly.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Visualize the dry 60/40 plate under UV light and then with iodine vapor in the same way.<strong> Note: </strong>To avoid side reactions, always check plates under UV light before exposing them to iodine.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Now, precisely copy the spots and the solvent fronts onto the corresponding drawings of the plates in your lab notebook. For each plate, measure and record the height of the solvent front relative to the starting line.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Lastly, measure the distance between the center of each spot and its starting line, and record it in your lab notebook. You can now move on to the next part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Unknown Compound Identification<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll use TLC to find out what&#39;s in an unknown analgesic. It could contain one or more of these compounds: aspirin, caffeine, and acetaminophen. These compounds are highly polar, so you&#39;ll use a premade mixture of 90% ethyl acetate and 10% hexane as the eluent.</p>
	<p>You&#39;ll run the three reference compounds and the unknown all on the same plate. Later, you&#39;ll use the Rf values of the reference compounds to identify the unknown. First, double-check which unknown material you were assigned to analyze before starting.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Now, lay a clean paper towel in the hood and obtain a new TLC plate. Prepare the top mark and the starting line the same way as before. Make four evenly spaced ticks on the plate, making sure that the outer marks are at least 0.5 cm away from the edges.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Draw an exact copy of the plate in your lab notebook and label the tick marks as &#8216;aspirin&#8217;, &#8216;acetaminophen&#8217;, &#8216;caffeine&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, bring your plate to the common bench. Spot solutions of aspirin, acetaminophen, caffeine, and the unknown that you were assigned on the appropriate marks using a clean capillary for each solution. <strong>Note: </strong>When you are spotting the unknown solution, don&#39;t dip the capillary too far into the solution vial to avoid clogging your capillary with insoluble material from the unknown substance.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Discard the capillaries and close the vials before bringing the plate back to your fume hood. Place the plate on a paper towel to finish drying.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Next, label a clean 50-mL screw-top jar &#8216;90% ethyl acetate, 10% hexane&#8217;, and bring it in a clean graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Place 2 &#8211; 3 mL of the 90/10 solution in the jar and return to your fume hood. Confirm that the solvent level will be below the spots on the plate. Then, place a clean filter paper upright in the jar and close it tightly.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the TLC plate is dry and the filter paper is saturated, carefully place the plate in the solvent. <strong>Note:</strong> It&#39;s essential to hold the plate level when you do this. If the solvent front is diagonal, the solvent distance traveled will be different for each spot, making it impossible to visually compare the unknown to the reference spots.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Wait for the solvent front to reach 1 cm from the top of the plate. Then, retrieve the plate, mark it solvent front, and let it dry while lying flat.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the plate is dry, visualize it with UV light and in the iodine chamber, and outline the spots in pencil.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">The unknown material could be a mixture of compounds, so look carefully for spots in line with all three reference spots.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, precisely copy the spots onto the drawing of the plate in your lab notebook. Measure and record the height of the solvent front and the spots relative to the starting line.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">When you are finished, retrieve the solvent-saturated filter papers from the jars and let them dry in the hood.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Pour leftover solvents into the standard organic waste, wash your glassware following your lab&#39;s usual methods, and put away your lab equipment.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Throw out used TLC plates in the lab trash or with glass waste, depending on the backing material.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Lastly, once the filters are dry, throw them out along with any remaining trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the R<sub>f</sub> values for phenol, benzoic acid, and butyl phenyl ether eluted with 100% hexane. For each compound on the TLC plate, measure the distance from the starting line to the center of its spot. Then, divide this value by the distance from the starting line to the solvent front.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Only butyl phenyl ether moved a measurable distance from the starting line, which is consistent with it being the least polar. For effective separation, the R<sub>f</sub> values should differ by 0.3 &#8211; 0.7. Thus, we can infer that phenol and benzoic acid are too polar to be separated effectively by an apolar solvent like pure hexane.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the R<sub>f</sub> values for the plate that you ran in a 60/40 mixture of hexane and ethyl acetate. Ethyl acetate is a moderately polar solvent, so the compounds traveled farther and are spaced farther apart on the plate.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">The separation order shows that benzoic acid, the most polar compound, had the highest affinity for silica gel, whereas butyl phenyl ether, the least polar compound, had the lowest affinity for silica gel. This is consistent with the assumption that less polar compounds travel farther on polar stationary phases.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Look at your third plate and calculate the R<sub>f</sub> values for aspirin, acetaminophen, and caffeine. Caffeine, the most polar complex of the three, traveled the least. Acetaminophen traveled the second farthest, while aspirin, the least polar, traveled the most.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Finally, identify the compounds in your unknown material by comparing its spot or spots to the three reference spots.</div>
		</li>
	</ul>
	</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Procedure

박층 크로마토그래피

Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Effects of Mobile Phase Composition on Compound ...

Synthesis of Luminol

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Synthesis of 3-Nitrophthalhydrazide<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll synthesize 3-aminophthalhydrazide, which is also called luminol, in a 2-step process. The first step is a condensation reaction between 3-nitrophthalic acid and hydrazine.</p>
	<p>During this step, the carboxylic acid groups are substituted with NH groups to produce 3-nitrophthalhydrazide and two water molecules. You must work in a fume hood throughout this lab because the reactions produce toxic vapors and gases.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. <strong>Note:</strong> Hydrazine is highly toxic and flammable, so avoid touching it and keep anything containing hydrazine closed or covered when you transport it between fume hoods.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Now measure 10 mL of deionized water and pour it into a 25-mL Erlenmeyer flask. Clamp the flask on a hot plate and place a thermometer in it.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Then, start heating the water. You&#39;ll want the temperature of the water to be 80 &#176;C, so set the hot plate setting to slightly higher.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">While it heats, set up a Bunsen burner under a clamp fixed on a lab stand.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Obtain a 25-mL test tube and adjust the clamp so that it will hold the test tube about 5 &#8211; 7 cm above the flame. Then, remove the test tube and place it in a 400-mL beaker.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Once the water reaches 80 &#176;C, adjust the heat setting to keep the temperature stable.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Then, weigh 0.6 g of 3-nitrophthalic acid in a tared weighing boat. Pour the 3-nitrophthalic acid into your test tube. Then, bring the test tube and a test tube stopper to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Use the provided volumetric pipette to carefully measure 0.9 mL of the 10% v/v hydrazine solution and transfer it to the test tube. Remember to cap the bottle of hydrazine solution and stopper your test tube before you return to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Clamp the test tube over the Bunsen burner and remove the stopper. Then, pour ~2 mL of triethylene glycol into a graduated cylinder and set it out of the way in your hood.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Now, add 2 or 3 boiling chips to the test tube. Place a high-temperature thermometer in the test tube and ignite the Bunsen burner. Heat the mixture with a medium flame until the 3-nitrophthalic acid dissolves.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Then, add the triethylene glycol and increase the heat of the flame. <strong>Note:</strong> Once the water from the hydrazine solution has boiled off, the temperature will continue increasing.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">When the reaction mixture reaches 210 &#176;C, turn down the heat and keep the mixture between 210 &#176;C and 220 &#176;C for 3 &#8211; 4 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Then, extinguish the flame and wait for the mixture to cool to 100 &#176;C.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Turn off the hotplate, remove the thermometer, and unclamp the flask. Use tongs to hold the flask and measure 8 mL of hot water with the graduated cylinder. Pour this hot water into the test tube and remove the thermometer.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Obtain a piece of plastic paraffin film and cover the mouth of the test tube. Then, use tongs to transfer the test tube to the holding beaker.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">At a lab sink, hold the test tube under running tap water to cool it, being careful not to let water into it.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Then, dry the outside of the tube and bring it back to your hood. Clamp the tube upright and remove the paraffin film. Let the yellow 3-nitrophthalhydrazide precipitate in the tube for 15 min.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">While you wait, add ~10 mL of deionized water to a 25-mL Erlenmeyer flask. Prepare an ice bath in a 250-mL beaker and start chilling the water in it.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Then, set up for vacuum filtration using a 250-mL filter flask, a B&#252;chner funnel, and a piece of circular filter paper.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Once the product has precipitated for at least 15 min, obtain a Pasteur pipette and wet the filter paper with a few drops of cold water.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Then, turn on the vacuum and pour the contents of the test tube into the B&#252;chner funnel. Rinse the remaining solid into the funnel with 1 &#8211; 2 mL of cold water.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Remove the boiling chips with tweezers and rinse them with a few drops of cold water to finish collecting the product.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Turn off the vacuum pump once no more liquid is dripping into the flask from the funnel.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Synthesis of 3-Aminopthalhydrazide<button class="expand">Expand</button>
	<p>In the second step of the luminol synthesis, you&#39;ll reduce the nitro group of 3-nitrophthalhydrazide using sodium dithionite in a basic solution of NaOH. This will produce 3-aminophthalhydrazide dianion.</p>
	<p>After the reduction, you&#39;ll add acetic acid to protonate the dianion, forming luminol. Sodium dithionite is highly reactive and degrades quickly, so you&#39;ll use a slight excess of it. After the reduction, you&#39;ll protonate the luminol dianion to decrease its solubility in water. Lastly, you&#39;ll precipitate and collect luminol as a yellow solid.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Place a clean 25-mL test tube in a beaker and use a clean spatula to transfer your 3-nitrophthalhydrazide to the test tube.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Set the B&#252;chner funnel aside and clamp the test tube over the Bunsen burner. Leave the filter flask in place for later.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, use a 10-mL graduated cylinder to obtain 3 mL of approximately 3 M NaOH. Pour the sodium hydroxide solution into the test tube and stir the mixture with a glass rod until the 3-nitrophthalhydrazide dissolves.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Now, weigh 2 g of sodium dithionite in a tared weighing boat and add it to the test tube. Obtain 3 mL of deionized water and pour it into the tube.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Stir the mixture well to dissolve as much of the solid as you can. Then, add a few clean boiling chips to the test tube and ignite the Bunsen burner.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Heat the mixture to boiling over a medium flame and then let it boil for 5 min.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">While you wait, measure 1.5 mL of glacial acetic acid and bring it to your hood.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Once the reaction mixture has boiled for 5 min, shut off the Bunsen burner and add the acetic acid to the tube. Let the mixture cool to room temperature, which usually takes 10 &#8211;15 minutes. Meanwhile, prepare an ice bath in a 600-mL beaker.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the mixture has cooled, place it in the ice bath and wait 15 min for the luminol to precipitate.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">Reassemble the vacuum filtration setup using a clean B&#252;chner funnel and a new piece of filter paper, and start cooling another 10 mL of deionized water in an ice bath.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Once the luminol has been in the ice bath for at least 15 min, wet the filter paper with cold water. Then, collect the solid luminol by vacuum filtration.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">Retrieve the boiling chips and rinse the luminol with more cold water. Once liquid stops dripping from the funnel, turn off the vacuum and transfer the luminol to a 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Now, prepare the combined filtrate for disposal. First, label a 250-mL beaker &#8216;hydrazine and dithionite waste&#8217;. Transfer the filtrate to the beaker and rinse the flask with deionized water to remove residual hydrazine and dithionite.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Next, dilute the filtrate with 10 mL of deionized water. Then, measure 10 mL of 1 M sodium carbonate and add it to the combined filtrates.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Bring a 50-mL graduated cylinder to the waste hood. Measure 40 mL of 10% sodium hypochlorite and pour it into the filtrate mixture. Place the beaker on the hot plate, add a stir bar, and secure a thermometer in the beaker.</div>
		</li>
		<li data-sub-note-item="2-18">
		<div class="lab-step">Heat the mixture to 50 &#176;C while stirring. The mixture must stay at 50 &#176;C for 1 h to oxidize leftover hydrazine and dithionite, so make sure that the temperature is stable and set a timer for 1 h before starting the last part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Chemiluminescence and&#160;Fluorescence<button class="expand">Expand</button>
	<p>For the last part of the lab, you&#39;ll mix some of the luminol you made with dimethyl sulfoxide over solid potassium hydroxide. The hydroxyl ions deprotonate the two amine groups. Then, ambient oxygen oxidizes the luminol dianion to 3-aminophthalate, or 3-APA, in an excited state. This unstable complex will quickly relax to the ground state, releasing energy as visible light. Since the excited 3-APA was the product of a chemical reaction, the emitted light is called chemiluminescence.</p>
	<p>Once you have seen the chemiluminescence from the oxidation reaction, you&#39;ll add fluorescein, a fluorescent molecule, to the mixture. Some excited 3-APA will transfer energy directly to fluorescein rather than emitting light, giving you a solution with two different light-emitting compounds.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-3">
		<div class="lab-step">Now, measure about 0.2 g of the luminol you made and place it in a 15-mL Erlenmeyer flask. It&#39;s OK if the solid is still damp.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Obtain 5 &#8211; 7 pellets of potassium hydroxide and pour them into the flask. Then, bring the flask, a stopper, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Measure 2 mL of DMSO, add it to the flask, and stopper it. Put the graduated cylinder in your fume hood and bring the flask to a dark room.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Shake the stoppered flask vigorously until you see chemiluminescence and then put the flask down. The light fades quickly because both the oxidation and relaxation are fast processes.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Shake the flask and let the light fade 6 &#8211; 10 more times without opening the flask. The light will be progressively dimmer as the oxygen in the flask is consumed. <strong>Note:</strong> To make it brighter, leave the flask open in your fume hood for a minute to replenish the oxygen.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Close the flask, return to the dark room, and shake the flask again to see the effect.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Now, measure 0.2 g of fluorescein dye and add it to your flask. Stopper the flask and return to the dark room. Shake the flask vigorously until the mixture is glowing brightly and then set it down to observe the difference in color from luminol alone.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">When you&#39;re finished, check on the filtrate. Once it has been at 50 &#176;C for at least 1 h, turn off the hotplate and wait for the beaker to cool to room temperature.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Then, add about 10 mL of tap water to the filtrate and flush it down the drain.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Dispose of the remaining chemical and glass waste in the appropriate containers, clean your glassware and tools, and put away your lab equipment.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<p>Under your oxidation reaction conditions, excited 3-APA emits blue-green light in a broad range around 500 nm when it relaxes, and fluorescein is excited by absorbing light at 480 &#8211; 490 nm and 515 &#8211; 525 nm.</p>
	<p>When you add fluorescein to the luminol oxidation reaction mixture, excited 3-APA can transfer energy that fluorescein would absorb as light directly to a nearby fluorescein molecule in a special interaction called nonradiative energy transfer. The resulting excited fluorescein emits yellow-green light when it relaxes.</p>
	<p>Excited 3-APA is highly unstable, so if no fluorescein is close enough for nonradiative energy transfer, it will relax by emitting light as usual. Thus, a spectrum of your final glowing mixture would show contributions from both blue-green and yellow-green light.</p>
	<ul class="lab-items">
	</ul>
	</li>
</ol>

Synthesis of Luminol and Chemiluminescence - Procedure

루미놀의 합성

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Synthesis of 3-NitrophthalhydrazideExpand
	In this lab, ...

Identification of Unknown Aldehydes and Ketones

<p>Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">DNPH Test for Aromatic Aldehydes and Ketones<button class="expand">Expand</button>
	<p>In this lab, you&#39;ll use the DNPH test, the Tollens&#39; test, and the iodoform test to identify two unknown aldehydes or ketones. You&#39;ll use butanone and benzaldehyde as known compounds to confirm that the tests are working as expected.</p>
	<p>You&#39;ll start the lab with the DNPH test, which relies on the reaction between 2,4-dinitrophenylhydrazine, or DNPH, and aldehydes and ketones. When you combine DNPH and an aldehyde or ketone in an acidic environment, they undergo a condensation reaction to form the corresponding hydrazone. The hydrazone then precipitates from solution. If the precipitate is red to orange, the aldehyde or ketone was aromatic. If it&#39;s orange to yellow, the aldehyde or ketone was non-aromatic or aliphatic.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on a lab coat, safety glasses, and nitrile gloves.<strong> Note: </strong>Many chemicals in this lab are toxic, corrosive, or flammable, so you will perform this lab in a fume hood. Change your gloves if you get any chemicals on them.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Get a vial of each known compound and choose two unknown compounds to analyze. Write the codes for your unknowns in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 250-mL beaker for aqueous waste and a 600-mL beaker for used pipettes. Put several clean Pasteur pipettes in another beaker and get more whenever you need them.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Obtain four small test tubes in a rack and label them with the four compound names, or codes, and the name of the first test.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Bring a 50-mL beaker and a graduated cylinder to the reagent hood and obtain 8 mL of the DNPH solution.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use four Pasteur pipettes to place 3 &#8211; 4 drops of each compound into its test tube. <strong>Note:</strong> Don&#39;t reuse pipettes during this lab because cross-contamination will invalidate the test results.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Add 2 mL of the DNPH solution to one of the test tubes and mix it well with a glass rod. Thoroughly rinse the glass rod with deionized water over your aqueous waste beaker to remove all traces of the ketone or aldehyde.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Add 2 mL of the DNPH solution to each of the other three test tubes in the same way. Make sure to thoroughly clean the glass rod after each one. When you&#39;re finished, let the solution sit for 15 min.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">While you wait, set up for vacuum filtration and fill a 250-mL beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">After 15 minutes, inspect the precipitates in the test tubes. <strong>Note:</strong> Benzaldehyde has an aromatic group, so you should see a red to orange solid in its test tube. Butanone is aliphatic, so you should see an orange to yellow solid in its test tube. Record the results in your lab notebook.
		<h4><strong>Table 1: DNPH, Tollens&#39;, and Iodoform Results</strong></h4>
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Compound</strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>DNPH </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Tollens&#39; </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Iodoform </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Solubility in water </strong></td>
					<td style="border: 1px solid; padding: 0px; text-align: center;"><strong>Hydrazone m.p. </strong></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Butanone</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
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					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">Benzaldehyde</td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
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					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
					<td style="border: 1px solid; padding: 0px;"></td>
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					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160; &#160;</td>
					<td style="border: 1px solid; padding: 0px;"></td>
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					<td style="border: 1px solid; padding: 0px;"></td>
					<td style="border: 1px solid; padding: 0px;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/69/Table_1._DNPH,_Tollens_,_and_Iodoform_results.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Compare the solids from the unknown compounds to the known compounds. For each unknown, record whether this solid was more red-orange, like benzaldehyde, or more orange-yellow, like butanone.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Use a clean Pasteur pipette to wet the filter paper of the vacuum filtration setup with deionized water and collect the solid from one of your unknowns by vacuum filtration. Rinse out any solid left in the tube with deionized water.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Wash the solid on the filter paper with 10 mL of deionized water.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Pick up a tiny amount of the solid with a clean glass rod and smear it on pH paper. If the pH is below 6.5, rinse the solid with another 10 mL of deionized water. Keep rinsing the solid and checking its pH until it is between 6.5 and 7.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Label a small watch glass for the unknown and carefully transfer the filter paper and solid to the watch glass to dry.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Thoroughly rinse the B&#252;chner funnel with deionized water, dispose of the filtrate in the DNPH solution waste, and reassemble the vacuum filtration setup with a new piece of filter paper.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Collect and wash the solids from the second unknown compound and from the two known compounds in the same way.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Leave the four solids in your fume hood to dry (1 h).</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">The Tollens&#8217; Test<button class="expand">Expand</button>
	<p>When you combine an aldehyde with diammine silver(I), or Tollens&#39; reagent, the reagent is reduced to metallic silver and ammonia. The silver either coats the inside of the reaction vessel or precipitates as a black solid, so it&#39;s easy to see that the reaction happened.</p>
	<p>Most ketones are harder to oxidize than aldehydes and therefore don&#39;t react with the Tollens&#39; reagent. For this lab, you can assume that if silver appears, the compound is an aldehyde. If silver does not appear, the compound is a ketone.</p>
	<p>The Tollens&#39; reagent solution contains a strong base and must be disposed of specially to avoid forming an explosive decomposition product. Handle it carefully and remember to put the Tollens&#39; test waste in the designated container.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place your used test tubes in a beaker and label four new tubes with the compound names or codes and Tollens&#39; test.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Bring a clean 50-mL beaker and a 5-mL graduated cylinder to the reagent hood and obtain 4 mL of Tollens&#39; reagent.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use clean pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Add 1 mL of Tollens&#39; reagent to each test tube and mix the solutions well with a clean glass rod, thoroughly rinsing the rod with deionized water between tubes. Set the rack of tubes to the side and let it sit for 10 min.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">While you wait, get about 100 mL of water in a 150-mL beaker and set it on a hotplate. Position a small 3-prong clamp above the beaker.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check the benzaldehyde tube after 10 min.<strong> Note:</strong> Benzaldehyde should have a positive result, as indicated by a silver film or precipitate. If you don&#39;t see a positive result in the benzaldehyde tube, secure a thermometer to the test tube and heat in the water bath at 35 &#176;C for 5 min. The silver film, or precipitate, should then appear. Record this result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Butanone won&#39;t react with Tollens&#39; reagent. <strong>Note: </strong>The butanone tube is an example of a negative Tollens&#39; test result. Record this in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Check the tubes of your unknowns. If you see a silver mirror or precipitate, record a positive Tollens&#39; test result for that unknown. If you don&#39;t, heat the tube in the water bath for 5 min and check again. If the test is still negative, record that result in your lab notebook.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">When you&#39;re done, turn off the hotplate and dispose of the Tollens&#39; test solutions in the appropriate waste container. Put silver-coated tubes in the collection box for acid cleaning and put the rest in your beaker.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Iodoform Test for Methyl Ketones<button class="expand">Expand</button>
	<p>When you combine iodine and a methyl ketone in aqueous base, iodine replaces the alpha methyl&#39;s hydrogens. An addition-elimination reaction followed by proton transfer yields iodoform and a carboxylate anion. Iodoform is insoluble in water, so you&#39;ll see a yellow precipitate if the compound that you&#39;re testing is a methyl ketone. If it is not a methyl ketone, the test solution will eventually turn brown because of the unreacted iodine.</p>
	<ul class="lab-items">
		<li data-sub-note-item="3-2">
		<div class="lab-step">Before the iodoform test, check the solubility of all four compounds in water. Label four clean test tubes with the compound names or codes and H<sub>2</sub>O. Label another four test tubes with the compound names and CHI<sub>3</sub>.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Use clean Pasteur pipettes to place 3 &#8211; 4 drops of each compound in its test tube.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">Add 2 mL of deionized water to each labeled tube. Mix well with a clean glass rod. Thoroughly clean the rod with deionized water between tubes.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Wait a few minutes for the liquids to settle before evaluating them. <strong>Note:</strong> Butanone is moderately soluble in water, so you should see little to no separation. Benzaldehyde is sparingly soluble in water, and its density is a little higher than water&#39;s. You should see a small layer at the bottom of the tube.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Inspect your unknowns for liquid layers and record the results for all four compounds in your lab notebook.</div>
		</li>
		<li data-sub-note-item="3-7">
		<div class="lab-step">Add 2 mL of deionized water to the butanone tube and any water-soluble ketones and mix them well with a clean glass rod. <strong>Note: </strong>If you have an unknown ketone that is insoluble in water, instead add 2 mL of 1,2-dimethoxyethane and mix it well with a clean glass rod.</div>
		</li>
		<li data-sub-note-item="3-8">
		<div class="lab-step">Now, perform the iodoform test, which is for ketones only. Use a clean 50-mL beaker and a small graduated cylinder to obtain 4 or 6 mL of 3 M NaOH, depending on whether you have two or three ketones to test.</div>
		</li>
		<li data-sub-note-item="3-9">
		<div class="lab-step">Pour ~10 mL of iodoform reagent into another 50-mL beaker.</div>
		</li>
		<li data-sub-note-item="3-10">
		<div class="lab-step">Add 2 mL of 3 M NaOH to each ketone tube and mix well, thoroughly cleaning the glass rod with deionized water between each tube.</div>
		</li>
		<li data-sub-note-item="3-11">
		<div class="lab-step">Obtain a clean Pasteur pipette and add the iodoform reagent dropwise to the butanone tube, frequently stirring until you see a yellow precipitate. <strong>Note:</strong> Butanone is a methyl ketone, so it will produce a positive result.</div>
		</li>
		<li data-sub-note-item="3-12">
		<div class="lab-step">Rinse the glass rod with deionized water and start adding the iodoform reagent dropwise to your first, or only, unknown ketone, while stirring frequently. Continue adding the reagent until either yellow iodoform precipitates, like you saw with butanone, or the solution stays brown, even after you stir it well. <strong>Note: </strong>The latter is a negative result.</div>
		</li>
		<li data-sub-note-item="3-13">
		<div class="lab-step">If you have another unknown ketone, clean the glass rod well and repeat the test with your second ketone.</div>
		</li>
		<li data-sub-note-item="3-14">
		<div class="lab-step">When you&#39;re finished, dispose of your iodine-containing solutions in the appropriate waste container.</div>
		</li>
		<li data-sub-note-item="3-15">
		<div class="lab-step">Once the solids from the DNPH test are dry, measure the melting point of each solid and record them in your lab notebook. Put the remaining solutes in the waste container for solid DNPH derivatives.</div>
		</li>
		<li data-sub-note-item="3-16">
		<div class="lab-step">Dispose of the leftover organic liquids and the aqueous aldehyde mixtures in the usual waste containers.</div>
		</li>
		<li data-sub-note-item="3-17">
		<div class="lab-step">Rinse the Pasteur pipettes used for the aldehydes and ketones with acetone before putting them in the glass waste. Pour your aqueous waste down the drain with running tap water. Clean your glassware, put away your lab equipment, and collect and throw out any trash in your fume hood before you leave.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="4" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="4-1">
		<div class="lab-step">Identify your unknown compounds. There are six options: acetophenone, butanal, 3-methyl-2-butanone, pentanal, 2-pentanone, and 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-2">
		<div class="lab-step">Look at the DNPH results. If the precipitate was red to orange, the unknown aldehyde or ketone is aromatic. If it was yellow, the unknown is aliphatic. <strong>Note:</strong> Since only one possible compound is aromatic, red precipitate means that the unknown was acetophenone. As confirmation, you should also have seen a negative Tollens&#39; test and a positive iodoform test.</div>
		</li>
		<li data-sub-note-item="4-3">
		<div class="lab-step">A yellow precipitate in the DNPH test means that the unknown could be any of the other five compounds.</div>
		</li>
		<li data-sub-note-item="4-4">
		<div class="lab-step">Look at the results of the Tollens&#39; test. If you saw a silver mirror or silver-gray precipitate in the test tube, the unknown compound is an aldehyde. If you didn&#39;t see any change, then the unknown compound is a ketone. If your unknown is an aldehyde, it could be pentanal or butanal.</div>
		</li>
		<li data-sub-note-item="4-5">
		<div class="lab-step">Compare the melting point of the unknown&#39;s hydrazone derivative to literature values for each aldehyde to figure out which one it was.</div>
		</li>
		<li data-sub-note-item="4-6">
		<div class="lab-step">If Tollens&#39; test was negative and the DNPH test showed an aliphatic compound, the unknown could be 3-methyl-2-butanone, 2-pentanone, or 3-pentanone.</div>
		</li>
		<li data-sub-note-item="4-7">
		<div class="lab-step">Check the results of the iodoform test to narrow it down. If you saw solid yellow iodoform, the compound was a methyl ketone. If you saw a brown solution, it was not a methyl ketone.</div>
		</li>
		<li data-sub-note-item="4-8">
		<div class="lab-step">Only one of the aliphatic ketones is not a methyl ketone, so if you saw a brown solution, the unknown was 3-pentanone. If you saw yellow iodoform precipitate from solution and the DNPH test also had yellow precipitate, then the unknown is 3-methyl-2-butanone or 2-pentanone.</div>
		</li>
		<li data-sub-note-item="4-9">
		<div class="lab-step">Use the melting point of the unknown&#39;s hydrazone derivative to figure out which methyl ketone you had.</div>
		</li>
	</ul>
	</li>
</ol>

Test for Aldehydes and Ketones: DNPH, Tollen's Test, and Iodoform Test - Procedure

미확인 알데히드(unknown aldehydes) 및 케톤(Ketones)의 확인

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	DNPH Test for Aromatic Aldehydes and KetonesExpand
	In ...